University of Veterinary Medicine Hannover

Institute of Virology

Department of Infectious Diseases

Biological characterization of

porcine pegivirus

THESIS

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY

(PhD)

awarded by the University of Veterinary Medicine Hannover

by

Johanna Kennedy

Koblenz

Hannover, Germany 2020

Supervisor Prof. Dr. Paul Becher

Supervision Group Prof. Dr. Paul Becher

Prof. Dr. Karl-Heinz Waldmann

Prof. Dr. Eike Steinmann

1st Evaluation Prof. Dr. Paul Becher

Institute of Virology, University of Veterinary Medicine

Hannover, Germany

Dr. Imke Steffen

Institute for Physiological Chemistry, University of Veterinary

Medicine Hannover, Germany

Prof. Dr. Eike Steinmann

Faculty of Medicine, Department of Molecular and Medical

Virology, Ruhr-University Bochum, Germany

2nd Evaluation Prof. Benedikt Kaufer, PhD

Institute of Virology, Free University of Berlin, Germany

Date of final exam 30th March 2020

Once again… for science, obviously.

Instant classic.

Parts of the thesis have been published previously in:

Research article

Kennedy J, Pfankuche VM, Hoeltig D, Postel A, Keuling O, Ciurkiewicz M,

Baumgärtner W, Becher P, Baechlein C. Genetic variability of porcine pegivirus in pigs

from Europe and China and insights into tissue tropism. Sci Rep. 2019 Jun 3;9(1):8174.

doi: 10.1038/s41598-019-44642-0

Poster and oral presentations

Kennedy J, Baechlein C, Hoeltig D, Becher P. Presence of porcine pegivirus in

domestic pigs and phylogenetic analysis of pegivirus strains from different parts of

the world. 10th Graduate school days, 2017, Bad Salzdetfurth, Germany.

Kennedy J, Baechlein C, Hoeltig D, Becher P. Presence of porcine pegivirus in

domestic pigs and phylogenetic analysis of pegivirus strains from different countries

in Europe and Asia. 28th Annual Meeting of the Society for Virology (GfV), 2018,

Würzburg, Germany.

Kennedy J, Pfankuche VM, Hoeltig D, Postel A, Keuling O, Ciurkiewicz M,

Baumgärtner W, Becher P, Baechlein C. Porcine pegivirus: genetic variability in pigs

from Europe and China, insights into tissue tropism and establishment of antibody

ELISA. 11th Graduate school days, 2018, Hannover, Germany.

Kennedy J, Pfankuche VM, Hoeltig D, Postel A, Ciurkiewicz M, Baumgärtner W,

Becher P, Baechlein C. Characterization of persistent pegivirus infection: serology,

transmission and replication in PBMCs. 29th Annual Meeting of the Society for

Virology (GfV), 2019, Düsseldorf, Germany.

Kennedy J, Pfankuche VM, Hoeltig D, Postel A, Ciurkiewicz M, Baumgärtner W,

Becher P, Baechlein C. Insights into porcine pegivirus infection: global distribution,

tissue tropism, and transmission. Keystone Symposia Conference on Positive-Strand

RNA Viruses, 2019, Killarney, Ireland.

Contents

I

Table of contents __________________________________________________________ I

List of abbreviations ______________________________________________________ III

List of figures ____________________________________________________________ VI

List of tables _____________________________________________________________VII

Table of contents

1 Introduction ___________________________________________________________ 1

1.1 Genus Pegivirus ____________________________________________________ 1

1.1.1 Discovery of pegiviruses _________________________________________ 1

1.1.2 Taxonomy _____________________________________________________ 2

1.1.3 Morphology and genome organization ____________________________ 4

1.1.4 Pegivirus protein functions _______________________________________ 5

1.2 Biology of pegivirus infection in pigs and other hosts ____________________ 6

1.2.1 Prevalence and seroprevalence ___________________________________ 6

1.2.2 Transmission __________________________________________________ 10

1.2.3 Persistence ____________________________________________________ 10

1.2.4 Tissue tropism _________________________________________________ 11

1.2.5 Co-infection with other pathogens and clinical relevance ____________ 12

1.3 Aims of the study __________________________________________________ 13

2 Genetic variability of porcine pegivirus in pigs from Europe and China and

insights into tissue tropism _________________________________________________ 15

Contents

II

3 Dissecting antibody reactivity and possible transmission routes in porcine

pegivirus infection ________________________________________________________ 35

4 Overall Discussion ____________________________________________________ 59

4.1 PPgV RNA detection in domestic pig serum samples from Europe and Asia _

_________________________________________________________________ 59

4.2 Phylogenetic analyses of PPgV ______________________________________ 60

4.3 No detection of PPgV RNA in wild boar ______________________________ 61

4.4 Persistent and transient PPgV infections ______________________________ 62

4.5 Investigation of PPgV tissue tropism _________________________________ 65

4.6 Insights into PPgV transmission routes _______________________________ 66

4.7 Antibody reactivity in Western blot and ELISA ________________________ 67

5 Summary ____________________________________________________________ 71

6 Zusammenfassung ____________________________________________________ 73

7 References ____________________________________________________________ 75

Contents

III

List of abbreviations

°C degrees Celcius

× g gravitational acceleration

µg microgram

µl microliter

µm micrometer

aa amino acid

Ab antibody

BPgV bat pegivirus

bp base pair

cDNA complementary deoxyribonucleic acid

Cq cycle quantification

CSFV classical swine fever virus

C-terminally carboxyl-terminally

E envelope protein

E2t carboxyl-terminally truncated envelope protein 2

E. coli Escherichia coli

ELISA enzyme-linked immunosorbent assay

ELISA100 ELISA coated with 100 ng protein per well

ELISA250 ELISA coated with 250 ng protein per well

EPgV equine pegivirus

FISH fluorescence in situ hybridization

FPLC fast protein liquid chromatography

GBV GB virus

h hour

HCV hepatitis C virus

HGV hepatitis G virus

HHPgV human hepegivirus

HIV human immunodeficiency virus

HPgV human pegivirus

HRP horseradish peroxidase

IgA immunoglobulin A

Contents

IV

IgG immunoglobulin G

IgM immunoglobulin M

IMAC immobilized metal ion chromatography

IPTG Isopropyl β-d-1-thiogalactopyranoside

IRES internal ribosome entry site

kb kilo base

kDa kilo Dalton

LB lysogeny broth

M molar

min minute

ml milliliter

mM millimolar

nm nanometer

no. number

NS non-structural protein

NS3h non-structural protein 3 helicase domain

nt nucleotide

N-terminal amino-terminal

NW New World

OD optical density

OW Old World

ORF open reading frame

PAGE polyacrylamide gel electrophoresis

PBS phosphate buffered saline

PBS-Tw phosphate buffered saline containing 0.05% Tween20

PCR polymerase chain reaction

PPgV porcine pegivirus

PVDF polyvinylidene difluoride

px protein x

qRT-PCR quantitative reverse transcription polymerase chain reaction

RdRp RNA-dependent RNA polymerase

RNA ribonucleic acid

RPgV rodent pegivirus

Contents

V

rpm revolutions per minute

RT room temperature

RT-PCR reverse transcription polymerase chain reaction

SDS sodium dodecyl sulfate

SPgV simian pegivirus

SPgVcpz simian pegivirus (chimpanzee)

ssRNA single-stranded RNA

TBS Tris-buffered saline

TDAV Theiler’s disease-associated virus

TierSchVersV Tierschutz-Versuchstierverordnung

TM TaqMan

TMB tetramethylbenzidine

UTR untranslated region

WB Western blot

Contents

VI

List of figures

Chapter 1

Figure 1-1. Phylogenetic relationship of pegivirus species A-K. __________________ 3

Figure 1-2. Predicted genome organization of porcine pegivirus. _________________ 5

Chapter 2

Figure 2-1. Phylogenetic analysis of porcine pegiviruses from different countries and

other mammalian pegiviruses. ______________________________________________ 21

Figure 2-2. Fluorescence in situ hybridization of porcine pegivirus (PPgV) positive

and negative pigs using a PPgV specific probe; overlay phase contrast and

immunofluorescence; bar = 100 µm. _________________________________________ 24

Chapter 3

Figure 3-1. Coomassie gel of NS3h protein before and after purification by IMAC. _ 45

Figure 3-2. Western blots of purified NS3h protein incubated with serum samples as

first antibody (Ab). ________________________________________________________ 46

Figure 3-3. Western blot of crude E2t protein incubated with serum samples that

showed NS3h-specific antibody (Ab) reactivity in Western blot and ELISA as first Ab.

_________________________________________________________________________ 48

Figure 3-4. PPgV viral genome quantity in serum (RNA positive results only) during

the course of infection in domestic pigs. ______________________________________ 50

Contents

VII

List of tables

Chapter 1

Table 1-1. Pegivirus species nomenclature and their respective hosts (Smith et al.,

2016). _____________________________________________________________________ 4

Chapter 2

Table 2-1. Porcine pegivirus genome detection rates and viral genome load in serum

samples from individual animals and herds from different countries in Europe and

Asia. ____________________________________________________________________ 19

Table 2-2. Number of pegivirus positive pigs of different age groups from Europe

and China. _______________________________________________________________ 19

Table 2-3. Porcine pegivirus RNA quantities and fluorescence in situ hybridization

results in blood and different tissues from two domestic pigs from Germany. _____ 22

Chapter 3

Table 3-1. Characterization of selected serum samples._________________________ 47

Chapter 1

1

1 Introduction

1.1 Genus Pegivirus

1.1.1 Discovery of pegiviruses

The genus Pegivirus owes its name to the history of the discovery of its first members

in 1995 (Simons et al., 1995b, Simons et al., 1995a, Linnen et al., 1996). The name was

first proposed by Stapleton and colleagues in 2011, consisting of two parts: “pe”

represents the characteristic of the viruses in frequently causing persistent infection in

their hosts, and “g” acknowledges the previous names “GB virus” and “hepatitis G

virus” (Stapleton et al., 2011). The first virus belonging to this group was discovered

in 1995 in tamarins and was identified as a primate virus with a flavivirus-like genome

closely related to the species Hepatitis C virus (HCV) (Simons et al., 1995b). Following

this, similar viruses were found in human sera by two independent working groups

who tentatively named them GB virus C (GBV-C) and hepatitis G virus (HGV) (Simons

et al., 1995a, Linnen et al., 1996). Though the newly discovered viruses were speculated

to be the causative agent of hepatitis for some time, studies failed to show a clear

association between virus infection and disease, rendering the name “hepatitis G

virus” misleading (Alter et al., 1997, Alter, 1997, Simons et al., 1995a, Mohr and

Stapleton, 2009, Theodore and Lemon, 1997). Further pegivirus species were identified

in the following years in a variety of mammalian hosts, including further primates,

bats, horses and rodents (Epstein et al., 2010, Chandriani et al., 2013, Kapoor et al.,

2013a, Quan et al., 2013, Firth et al., 2014, Kapoor et al., 2013b). In 2016, pegivirus

sequences were discovered in serum samples of domestic pigs from Germany, and the

newly discovered virus species was designated as Porcine pegivirus (PPgV) (Baechlein

et al., 2016).

Chapter 1

2

1.1.2 Taxonomy

The taxonomy within the Flaviviridae family was recently updated based on

phylogenetic relationships and virus characteristics to include newly discovered virus

species, which lead to the addition of the genus Pegivirus (Smith et al., 2016, Simmonds

et al., 2017, Adams et al., 2013). Highly significant human pathogens belong to this

family, including HCV in the Hepacivirus genus, and Yellow fever virus, dengue virus

and West Nile virus in the Flavivirus genus. Economically important animal pathogens

like bovine viral diarrhea virus, classical swine fever virus (CSFV) and border disease

virus of sheep belong to the Pestivirus genus (Simmonds et al., 2017). In contrast to the

major pathogens found within the first three genera of the Flaviviridae family, none of

the pegivirus species have been clearly associated with causing disease in their hosts

(Smith et al., 2016).

The genus Pegivirus currently contains eleven species, Pegivirus A-K, that infect a

variety of mammalian hosts, as shown in Figure 1-1 and Table 1-1 (Simons et al., 1995b,

Simons et al., 1995a, Linnen et al., 1996, Epstein et al., 2010, Quan et al., 2013,

Chandriani et al., 2013, Kapoor et al., 2013a, Kapoor et al., 2013b, Firth et al., 2014,

Baechlein et al., 2016). The type species of Pegivirus K is represented by “PPgV_903”,

the full-length viral genome sequence of which was isolated from serum of a domestic

pig from Germany (Baechlein et al., 2016, Smith et al., 2016).

Chapter 1

3

Figure 1-1. Phylogenetic relationship of pegivirus species A-K. The amino acid

multiple sequence alignment of the complete coding region of the respective viruses

was performed with ClustalW in BioEdit 7.0 (Hall, 1999). The Maximum Likelihood

phylogenetic tree was calculated with MEGA X (Kumar et al., 2018) using the Le and

Gascuel model (Le and Gascuel, 2008) with frequencies and a gamma distribution of

variation with invariant sites. Analysis was performed with 100 bootstrap replicates

(Felsenstein, 1985) and numbers along branches represent the percentage bootstrap

values. The scale bar indicates substitutions per site. GenBank accession numbers are

in parentheses.

Chapter 1

4

Table 1-1. Pegivirus species nomenclature and their respective hosts (Smith et al.,

2016).

Species Hosts Previous / other names Abbreviation

Pegivirus A NW primate

& OW bat GB virus A

SPgV

BPgV

Pegivirus B OW bat GB virus D BPgV

Pegivirus C human

& OW primate

GB virus C

hepatitis G virus

HPgV

SPgVcpz

Pegivirus D horse Theiler’s disease-associated

virus TDAV

Pegivirus E horse equine pegivirus EPgV

Pegivirus F NW bat bat pegivirus BPgV

Pegivirus G OW bat bat pegivirus BPgV

Pegivirus H human human hepegivirus

human pegivirus 2 HHPgV

Pegivirus I NW bat bat pegivirus BPgV

Pegivirus J NW rodent rodent pegivirus RPgV

Pegivirus K pig porcine pegivirus PPgV

NW, New World; OW, Old World

1.1.3 Morphology and genome organization

Pegiviruses are enveloped viruses with a spherical virion of 60-70 nm in size (Xiang et

al., 1999). In contrast to other members of the family Flaviviridae, pegiviruses appear to

have a truncated core-coding region or absence thereof, though biochemical

characterization and electron microscopy of human pegivirus (HPgV) suggest it has a

capsid of uncertain origin (Xiang et al., 1998, Xiang et al., 1999).

The positive-sense, single-stranded RNA genome of pegiviruses is non-segmented and

ranges in size from 8.9-11.3 kilo bases (kb). It contains a single open reading frame

(ORF), which is flanked by 5’ and 3’ untranslated regions (UTRs) (Simmonds et al.,

2017). In contrast to the flavivirus 5’ UTR, which contains a type I cap, the 5’ UTRs of

hepaciviruses, pestiviruses and pegiviruses possess an internal ribosome entry site

(IRES) for translation initiation (Simmonds et al., 2017, Simons et al., 1996). Pegivirus

IRES elements are structurally similar to the type I IRES of picornaviruses, or to the

Chapter 1

5

type IV IRES elements (Pegivirus H, J and F) seen in hepaci- and pestiviruses, though

in both cases sequence identity is limited (Quan et al., 2013, Kapoor et al., 2015). As is

common in hepaciviruses, BPgVs belonging to Pegivirus F contain a micro RNA-122

binding site in their 5’ UTR, while such sites are lacking in other pegiviruses (Smith et

al., 2016). Comparison of PPgV sequences evidences high amino acid (aa) identities

indicative of conserved genome regions within the putative non-structural protein 3

(NS3) and non-structural protein (NS) 5B coding regions, which coincides with

findings when comparing HPgV and HCV (Baechlein et al., 2016, Leary et al., 1996b).

1.1.4 Pegivirus protein functions

The ORF of members of the Flaviviridae family is translated into a large polyprotein

that is co- and post-translationally cleaved by cellular- and viral proteases (Figure 1-2).

As mentioned above, the origin of the pegivirus capsid has not been determined, as a

core-coding region like that seen in other Flaviviridae members appears to be lacking

in most pegiviruses, including PPgV (Mohr and Stapleton, 2009, Simons et al., 1996,

Baechlein et al., 2016). Most pegivirus protein functions have not been studied in detail

and predicted functions are mostly inferred from sequence comparison with

homologous proteins within the hepaciviruses, mainly HCV (Simmonds et al., 2017,

Mohr and Stapleton, 2009).

Figure 1-2. Predicted genome organization of porcine pegivirus. Schema was newly

constructed for this thesis modified from (Baechlein et al., 2016). Amino acid (aa) sizes

of the individual predicted mature proteins are indicated below. The predicted

cleavage sites are shown by grey (cellular signal peptidases) and black triangles (viral

proteases).

Chapter 1

6

Like hepaciviruses, pegiviruses have two predicted envelope glycoproteins (E), E1 and

E2, which appear to be inserted into the viral envelope in the form of heterodimers

(Mohr and Stapleton, 2009). E1 and E2 of PPgV have four N-X-S/T glycosylation sites

each, while the number of such sites varies from three to eleven in other pegivirus

species (Smith et al., 2016). Protein x (px), the homologue to p7 in HCV, differs in size

within the pegivirus species (Mohr and Stapleton, 2009).

Of the predicted six non-structural proteins of pegiviruses, NS2 is thought to be

involved in the cleavage of NS2-NS3 as an autoprotease, as it is seen in HCV. The

predicted function of HPgV NS3 is that of a viral helicase and of a chymotrypsin-like

serine protease, which is responsible for the cleavage of the remaining NS proteins and

uses NS4A as a co-factor, a function that is also thought to occur in other pegivirus

species (Epstein et al., 2010, Major and Feinstone, 1997, Mohr and Stapleton, 2009,

Moradpour et al., 2007, Robertson, 2001, Leary et al., 1996b, Stapleton, 2003). NS5A

appears to be an interferone sensitivity-determining region, and the predicted function

of NS5B is that of an RNA-dependent RNA polymerase (RdRp) (Leary et al., 1996b,

Stapleton, 2003, Linnen et al., 1996, Simons et al., 2000).

1.2 Biology of pegivirus infection in pigs and other hosts

1.2.1 Prevalence and seroprevalence

Porcine pegivirus RNA detection methods and rates

PPgV was first discovered in serum of domestic pigs from Germany using high-

throughput sequencing methods. It was subsequently detected in 10 of 455 (2.2%)

serum samples from 10 of 37 (27.0%) pig holdings by SYBR-Green-based quantitative

reverse transcription polymerase chain reaction (qRT-PCR) targeting NS3 (Baechlein

et al., 2016). Following this, Yang and colleagues used conventional RT-PCR, which

targets a conserved region in NS5B, to screen 159 porcine serum samples from 15 US

Chapter 1

7

states, of which 24 (15.1%, from 10 US states) were PPgV RNA positive, evidencing a

much higher detection rate than that previously found in Germany (Yang et al., 2018).

A study from China reported the detection of PPgV genome in 34 of 469 (7.3%) porcine

sera using nested RT-PCR targeting NS3, and observed an ascending detection rate

from suckling piglets (1.6%) to nursing piglets (1.9%), finishing pigs (6.6%), and sows

(11.3%) (Lei et al., 2019).

Human pegivirus RNA and antibody prevalence

HPgV (Pegivirus C) is distributed globally and an estimated 750 million people are

viremic, while another 750 million to 2 billion people have evidence of prior HPgV

infection. It is thus possibly the most prevalent human RNA virus causing persistent

infection, and a major contributor to the human virome (Stapleton et al., 2014,

Stapleton, 2003, Chivero and Stapleton, 2015).

Antibodies (Abs) against HPgV are usually detected after clearance of viremia, thus

exposure rates are calculated as the sum of RNA positive and Ab positive rates (Tacke

et al., 1997, Gutierrez et al., 1997, Thomas et al., 1998). The prevalence of HPgV viremia

in healthy blood donors from developed countries is 1-5%, and another 5-20% of

individuals have anti-E2 Abs, leading to a total exposure rate between 6 and 25%

(Mohr and Stapleton, 2009, Stapleton et al., 2011, Blair et al., 1998, Gutierrez et al., 1997,

Pilot-Matias et al., 1996a, Tacke et al., 1997). Rates are higher in blood donors from

developing countries, reaching close to 20% RNA detection rate in some regions (Mohr

and Stapleton, 2009, Polgreen et al., 2003). However, prevalence of viremia is

significantly higher in high-risk groups, namely individuals with coexistent blood-

borne or sexually transmitted infections, and nearly universal exposure is

demonstrated in some populations, such as intravenous drug users and human

immunodeficiency virus (HIV)-positive men who have sex with men (Alter, 1997,

Theodore and Lemon, 1997, Stapleton, 2003, Scallan et al., 1998, Stapleton et al., 2011,

Chapter 1

8

Williams et al., 2004, Dawson et al., 1996, Gutierrez et al., 1997, Schlauder et al., 1995,

Xiang et al., 2001).

Recently, Pegivirus H, a further pegivirus species infecting humans, was identified

(Berg et al., 2015, Kapoor et al., 2015). Prevalence of human hepegivirus (HHPgV, also

previously named human pegivirus 2) infection is much lower than that of HPgV, with

evidence of exposure to HHPgV (RNA and Ab) detected in 0.45-1.33% of cases (Berg

et al., 2015, Coller et al., 2016, Kapoor et al., 2015). In contrast to HPgV, Abs against

HHPgV were frequently detected during viremia (Berg et al., 2015, Coller et al., 2016).

Characteristics and detection methods of HPgV-specific antibodies

While various HPgV proteins have been used for Ab detection by expression in

Escherichia coli (E. coli), mammalian expressed, C-terminally truncated E2 was

identified as a useful antigen for studying HPgV exposure (Dawson et al., 1996, Pilot-

Matias et al., 1996b, Pilot-Matias et al., 1996a, Dille et al., 1997).

Anti-HPgV Ab development is usually restricted to conformation-dependent anti-E2

Abs that develop after clearance of viremia, as described above (Gutierrez et al., 1997,

Tacke et al., 1997, Tanaka et al., 1998, Thomas et al., 1998). Anti-E2 antibodies are long-

lived and provide a certain degree of protection from reinfection, indicating

neutralizing activity (Tillmann et al., 1998, Elkayam et al., 1999, Gutierrez et al., 1997).

Additionally, some studies have described the detection of anti-HPgV peptide

reactivity during viremia; however, Ab development is restricted to E2 in most cases,

suggesting that the E2 antigenic site is immunodominant in humans (McLinden et al.,

2006, Pilot-Matias et al., 1996b, Fernandez-Vidal et al., 2007, Gomara et al., 2010,

Schwarze-Zander et al., 2006, Tan et al., 1999, Van der Bij et al., 2005, Xiang et al., 1998).

Moreover, HHPgV-specific Abs have been detected using mammalian expressed E2,

as well as bacterially expressed NS4A/B and additional peptides located to NS3,

NS4A/B and NS5B (Berg et al., 2015, Coller et al., 2016).

Chapter 1

9

Pegivirus RNA and antibody detection rates in other mammals

Equine pegivirus (EPgV, Pegivirus E) RNA has been detected in horse serum samples

from the United States (9.5%), Brazil (0.8-14.2%), China (1.1%), Germany (13.4%) and

England, Scotland and France (3.6%) (Kapoor et al., 2013a, de Souza et al., 2015,

Figueiredo et al., 2019, Lu et al., 2018, Lyons et al., 2014, Postel et al., 2016). Theiler’s

disease-associated virus (TDAV, Pegivirus D) was first detected in 16 horses from the

United States: one was the donor horse of TDAV-containing antitoxin serum and the

other 15 horses had been exposed to this serum (Chandriani et al., 2013). Since then

TDAV has only been found in horses from Brazil in one study (1.6%), while two other

studies failed to find TDAV RNA in 114 and 177 samples from Brazil and China,

respectively (Figueiredo et al., 2019, de Souza et al., 2015, Lu et al., 2016).

For detection of anti-EPgV Abs, the NS3 helicase domain (NS3h) was expressed in E.

coli and crude NS3h-containing bacterial lysates were used in indirect ELISA. Of 328

horses from Scotland, England and France, 218 (66.5%) were positive for Ab in ELISA

and 88.5% of those were confirmed by Western blot with the same protein. Among

these, of the 12 horses with active EPgV infection (3.7%), 10 were also Ab positive

(Lyons et al., 2014).

BPgV was initially detected in 5% (5 of 98) of free ranging bats from Bangladesh

(Epstein et al., 2010), and further BPgV species were identified in 4% of 1,615 animals

belonging to 21 species of New World and Old World bats (Quan et al., 2013). Simian

pegiviruses (SPgV) have been found in various Sanguinus, Callithrix, and Aotus species

(Bukh and Apgar, 1997, Leary et al., 1996a, Muerhoff et al., 1995, Simons et al., 1995b).

In addition, viruses belonging to Pegivirus C naturally infect chimpanzees (SPgVcpz),

sequences of which form a separate phylogenetic group to HPgVs (see also Figure 1-1)

(Adams et al., 1998, Birkenmeyer et al., 1998).

Chapter 1

10

1.2.2 Transmission

HPgV has been shown to be transmitted by exposure to infected blood, sexually, and

vertically from mother to child (Bhanich Supapol et al., 2009, Hino et al., 1998,

Kleinman, 2001, Lin et al., 1998, Ohto et al., 2000, Stapleton, 2003). These transmission

routes are largely comparable with those found in HIV, explaining common HIV-

HPgV co-infection, and high-risk groups (i.e. intravenous drug users), as mentioned

above (Stapleton, 2003). Sexual transmission of HPgV is much more efficient than that

of HCV (Lauer and Walker, 2001, Sarrazin et al., 2000, Scallan et al., 1998, Nerurkar et

al., 1998, Bourlet et al., 1999, Hollingsworth et al., 1998, Xiang et al., 2001).

TDAV was shown to be transmitted to healthy horses by experimental intravenous

inoculation of antitoxin horse serum containing TDAV (Chandriani et al., 2013). EPgV

and TDAV were detected in commercially available equine serum pools from various

countries, indicating that transmission by products containing equine serum may be

possible (Postel et al., 2016).

The transmission of bat pegiviruses has not been studied in detail, however, BPgV

genome was detectable in saliva and rectal swabs, but not in urine of bats, indicating

that horizontal transmission by shedding in excreta or during fighting, grooming, or

sharing of food may be possible (Epstein et al., 2010, Quan et al., 2013). Studies on the

transmission of PPgV between pigs have not been reported to date.

1.2.3 Persistence

As RNA viruses, HPgV and HCV are unusual in frequently causing persistent

infections in immunocompetent hosts (Chivero and Stapleton, 2015). Though HPgV

persistence is not as frequent as that seen in HCV, it is found in roughly 25% of cases,

while the other 75% of infections are cleared within two years (Gutierrez et al., 1997,

Tanaka et al., 1998). HPgV infection can be long-lived – it was documented for 16 years

in one individual (Masuko et al., 1996) – and during persistence the viral load usually

Chapter 1

11

remains constant (Lefrere et al., 1999). Many aspects of immune evasion by RNA

viruses, including hepaciviruses and pegiviruses, remain poorly understood (Chivero

and Stapleton, 2015). Hypervariable regions of the E2 protein, mutation of

immunodominant T-cell epitopes, and chronic stimulation of T-cells leading to T-cell

exhaustion are mechanisms that have been proposed for HCV immune evasion, but

apparently do not apply to HPgV (Keck et al., 2009, Burke and Cox, 2010, Mohr and

Stapleton, 2009, Lemon, 2010, Stapleton et al., 2012). Persistence was also observed in

HHPgV infection (Kapoor et al., 2015, Berg et al., 2015), as was life-long SPgV infection

(Simons et al., 1995b). Persistent PPgV infection was observed in three pigs from

Germany, in which viral RNA was detected for 7, 16 and 22 months (Baechlein, 2016),

but has not been studied in greater depth.

1.2.4 Tissue tropism

The tissue tropism of most pegivirus species, including PPgV, has not been assessed

in detail. Though HPgV was speculated to be a causative agent of hepatitis for some

years, clear evidence of a causal association with hepatitis and evidence of replication

in liver tissue, such as viral negative-strand RNA as a replication intermediate, were

lacking or inconclusive (Chivero and Stapleton, 2015, Fan et al., 1999, Pessoa et al.,

1998, Tucker et al., 2000, Berg et al., 1999, Laskus et al., 1997, Laras et al., 1999).

Compared with HCV, a hepatotropic virus in which viral RNA levels are higher in

liver than serum, the opposite is the case for HPgV (Pessoa et al., 1998, Chivero and

Stapleton, 2015, Manns et al., 2017). Rather, HPgV negative-strand RNA has been

detected in bone marrow and spleen of infected individuals (Radkowski et al., 2000,

Tucker et al., 2000) and further evidence suggested that HPgV is lymphotropic, which

has also been demonstrated to be the case for SPgV (Fogeda et al., 1999, George et al.,

2006, Xiang et al., 2000, Kobayashi et al., 1999, Laskus et al., 1998, Stapleton et al., 2011).

HPgV replicates ex vivo in peripheral blood mononuclear cells (PBMCs) isolated from

HPgV RNA positive individuals (Fogeda et al., 1999, George et al., 2003, Rydze et al.,

Chapter 1

12

2012). PBMCs can also be infected in vitro using serum-derived HPgV (Chivero et al.,

2014, Xiang et al., 2000). Viral RNA was detected in natural killer cells, monocytes and

diverse subsets of T- and B-lymphocytes, including naïve, central memory and effector

memory T-cells, leading to the suggestion that HPgV may infect hematopoietic

precursor cells, which maintain infection during differentiation (Chivero and

Stapleton, 2015, Chivero et al., 2014). However, the primary permissive cell type(s) for

pegiviruses remain unknown (Chivero and Stapleton, 2015).

1.2.5 Co-infection with other pathogens and clinical relevance

Co-infection of HPgV and HIV in humans

As mentioned above, due to high similarity in transmission routes, HPgV and HIV co-

infection is common (Stapleton, 2003). Several studies have shown beneficial effects on

the outcome of HIV-infection attributed to HPgV, including longer survival in co-

infected individuals (Heringlake et al., 1998, Tillmann et al., 2001, Nunnari et al., 2003,

Toyoda et al., 1998, Williams et al., 2004, Xiang et al., 2001, Zhang et al., 2006, Vahidnia

et al., 2012). Such effects caused by HPgV can be explained by various mechanisms,

including alterations of cytokine profile, modification of HIV co-receptor expression,

direct inhibition of HIV entry through HPgV E2, and modulation of T-cell activation,

among others (Schwarze-Zander et al., 2012, Nunnari et al., 2003, Capobianchi et al.,

2006, Chang et al., 2007, Haro et al., 2011, Herrera et al., 2009, Herrera et al., 2010, Jung

et al., 2007, Koedel et al., 2011, Mohr et al., 2010).

Clinical relevance of pegivirus infection in humans

Several studies have suggested an association between HPgV infection and an

increased risk of non-Hodgkin lymphoma (NHL), attributing a possible etiologic role

in the development of NHL to chronic immune stimulation or impaired

immunosurveillance, to HPgV (Chang et al., 2014, Civardi et al., 1998, Collier et al.,

Chapter 1

13

1999, De Renzo et al., 2002, Giannoulis et al., 2004, Kaya et al., 2002, Minton et al., 1998,

Zignego et al., 1997, Ellenrieder et al., 1998, Keresztes et al., 2003, Michaelis et al., 2003,

Nakamura et al., 1997, Krajden et al., 2010). Furthermore, HPgV RNA has been

detected in brain tissue and in cerebrospinal fluid of individuals with encephalitis of

unknown cause, although no causal association has been shown (Kriesel et al., 2012,

Fridholm et al., 2016, Balcom et al., 2018, Tuddenham et al., 2019).

Disease association in animals

TDAV was suggested as the causative agent of a Theiler’s disease outbreak in horses

in the United States, but recent studies indicate that a newly discovered member of the

copiparvoviruses, namely equine parvovirus hepatitis, is responsible for acute serum

hepatitis in horses (Chandriani et al., 2013, Divers et al., 2018).

To date, infection with PPgV has not been shown to cause any disease in pigs, and

viral RNA can be detected in apparently healthy animals (Baechlein et al., 2016). One

study detected PPgV in a serum sample from a farm with pigs exhibiting lameness

and vesicles in the United States, but porcine parvovirus and astrovirus were also

detected. In the same study, nine vesicular swab samples that were additionally tested

for PPgV presence were found negative (Yang et al., 2018).

Disease association is similarly unknown for other pegiviruses and they are

considered as apathogenic to date. Further research is necessary to better understand

clinical implications of pegivirus infection in animal hosts and humans.

1.3 Aims of the study

Since the first description of PPgV in domestic pigs from Germany in 2016, only a

handful of studies have been published, which described mainly PPgV genome

detection in pigs from the United States and China (Baechlein et al., 2016, Chen et al.,

2019, Lei et al., 2019, Li et al., 2019, Yang et al., 2018). Many aspects of pegivirus

Chapter 1

14

infection, even in human hosts, remain elusive, not least due to poor in vitro replication

and the lack of an animal model in the case of HPgV (Chivero and Stapleton, 2015).

The overall objective of this project was to further biologically characterize porcine

pegivirus, specifically including method development to allow insights into RNA and

antibody prevalence, tissue tropism and sites of viral replication, host range,

transmission routes, and antibody response. In addition to method development, the

acquisition of blood, tissue and excretion samples from PPgV-infected pigs was an

essential step in the furtherance of this project.

The first aim of the project was the establishment and validation of a TaqMan-based

qRT-PCR and the development of an in vitro-transcribed RNA copy standard to allow

accurate quantification of PPgV RNA isolated from serum, tissues and excretion

samples. The second objective was the establishment of a nested PCR for the

amplification of a genome region suitable for sequencing that permitted subsequent

phylogenetic analyses. A further goal was the expression of PPgV proteins, namely of

the NS3 helicase domain (NS3h), and of truncated E2 (E2t), to evaluate these as

possible markers of past or present PPgV infection and, as such, as possible candidates

in antibody detection assays.

The development of these methods allows the investigation of various aspects of PPgV

biology, including virus distribution and spread, tissue tropism, transmission routes,

and infection dynamics, among others. The understanding of such aspects is essential

for examining not only how PPgV may influence porcine health, but also in

determining whether PPgV infection in the porcine host may be a valuable asset in the

study of HPgV infection in humans.

Chapter 2

15

2 Genetic variability of porcine pegivirus in pigs from Europe and

China and insights into tissue tropism

Kennedy J, Pfankuche VM, Hoeltig D, Postel A, Keuling O, Ciurkiewicz M,

Baumgärtner W, Becher P, Baechlein C.

This chapter was published in Scientific Reports journal

Kennedy J, Pfankuche VM, Hoeltig D, Postel A, Keuling O, Ciurkiewicz M,

Baumgärtner W, Becher P, Baechlein C. Genetic variability of porcine pegivirus in pigs

from Europe and China and insights into tissue tropism. Sci Rep. 2019 Jun 3;9(1):8174.

doi: 10.1038/s41598-019-44642-0

Contribution as first author:

Experimental work: Establishment and optimization of TM qRT-PCR for screening of

the presence of PPgV RNA in serum and tissue samples, establishment and

optimization of sequencing RT-PCR for phylogenetic analyses, sample preparation,

genome amplification, submission for sequencing. Evaluation and scientific

presentation of the results: Analyses and graphical presentation of qRT-PCR and

sequencing data, performing phylogenetic analyses. Scientific writing: preparation of

the manuscript, tables and figure (phylogenetic tree).

Chapter 2

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Abstract

Pegiviruses belong to the family Flaviviridae and have been found in humans and other

mammalian species. To date eleven different pegivirus species (Pegivirus A-K) have

been described. However, little is known about the tissue tropism and replication of

pegiviruses. In 2016, a so far unknown porcine pegivirus (PPgV, Pegivirus K) was

described and persistent infection in the host, similar to human pegivirus, was

reported. In this study, qRT-PCR, phylogenetic analyses and fluorescence in situ

hybridization (FISH) were implemented to detect and quantify PPgV genome content

in serum samples from domestic pigs from Europe and Asia, in tissue and peripheral

blood mononuclear cell (PBMC) samples and wild boar serum samples from Germany.

PPgV was detectable in 2.7% of investigated domestic pigs from Europe and China

(viral genome load 2.4 × 102 to 2.0 × 106 PPgV copies/ml), while all wild boar samples

were tested negative. Phylogenetic analyses revealed pairwise nucleotide identities

>90% among PPgVs. Finally, PPgV was detected in liver, thymus and PBMCs by qRT-

PCR and FISH, suggesting liver- and lymphotropism. Taken together, this study

provides first insights into the tissue tropism of PPgV and shows its distribution and

genetic variability in Europe and China.

Chapter 2

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Introduction

Pegiviruses comprise a group of positive-sense, single-stranded RNA viruses, with a

genome size of 9–13 kb, that were recently classified into eleven species (Pegivirus A-

K) within the genus Pegivirus in the Flaviviridae family1. They can infect humans as well

as a range of mammalian species, including primates, bats, rodents, horses and pigs2–9.

While pegiviruses are known to cause persistent infections in humans and horses, their

pathogenicity remains largely unknown1,4,10–12. Though a pegivirus was identified in

horses with Theiler’s Disease in the USA5, recent studies imply that viruses of the

copiparvovirus group are associated with serum hepatitis in horses13,14. Human

pegiviruses (HPgV) are distributed globally and viral RNA is present in roughly 750

million people, making it one of the most prevalent human RNA viruses15.

Though HPgV was initially thought to be hepatotropic and a possible agent of Non-

A-E hepatitis, evidence of viral replication in the liver of infected patients is missing

or inconclusive16–18. Rather, as HPgV replication has been shown in peripheral blood

mononuclear cells (PBMCs) ex vivo for several weeks, the virus appears to be

lymphotropic19–21. Additionally, HPgV RNA has been found in serum microvesicles,

which have successfully delivered viral RNA to uninfected PBMCs that then

supported HPgV replication ex vivo22. Interestingly, pegivirus infection in humans may

have a beneficial effect on the outcome of human immunodeficiency virus type 1

(HIV-1) infections in individuals co-infected with both viruses, including reduced

retroviral loads, slower progression to AIDS and improved survival rates. These

benefits are attributed to immune-modulating effects as well as direct and indirect

antagonistic mechanisms of HPgV on HIV-1 infection23.

Porcine pegiviruses (PPgV) were first described in domestic pigs from Germany in

20169. The study reported a PPgV detection rate of 2.2% (10 of 455) in porcine serum

samples and described persistent infection for up to 22 months in three pigs that did

Chapter 2

18

not display any clinical signs of disease. Apart from Germany, presence of PPgV has

been investigated in North America, where a recent study revealed a PPgV detection

rate of 15.1% (24 of 159 samples) in the USA24. Additionally, a recent study investigated

469 porcine serum samples from China, 34 (7.25%) of which were found PPgV positive.

Samples originated from different age groups and proved an ascending trend in the

PPgV positive rate from suckling piglets (1.61%) and nursing piglets (1.85%) to

finishing pigs (6.56%) and sows (11.34%)25.

In this study we analyzed the presence of PPgV genome in pigs from Europe and Asia.

To clarify whether wild boar might play a role in the epidemiology of PPgV, as seen

in infections with, for example, classical swine fever virus26,27, African swine fever

virus28 and atypical porcine pestivirus (APPV)29, we also investigated the presence of

PPgV genome in wild boar serum samples from Germany. To date the primary

permissive cell type(s) of HPgV and other pegiviruses remain unknown. For this

reason, we analyzed the tissue and cell tropism of PPgV through detection and

quantification of viral RNA in tissues and PBMCs from PPgV positive pigs using qRT-

PCR and fluorescence in situ hybridization (FISH).

Results

PPgV RNA in serum samples from Europe and Asia. The in vitro transcribed RNA

copy standard evidenced a highly efficient qRT-PCR assay that was able to detect ten

viral genome copies per reaction at Cq values around 36. PPgV genome was detectable

in 47 of 1,736 (2.7%) serum samples from domestic pigs corresponding to 20 out of 132

herds (15.2%) (Table 2-1). Highest detection rates were found in individual animals

from Great Britain (10.3%) and in herds from China (58.3%). In the different age groups

investigated here, the PPgV positive rates were 1.9% in animals under 4 weeks of age,

1.2% in fattening pigs over 4 weeks of age, 3.4% in sows and boars, and 10.1% in pigs

of unknown age and use (Table 2-2). Viral loads varied between 2.4 × 102 and 2.0 × 106

Chapter 2

19

PPgV RNA copies/ml serum, with an overall average of 3.8 × 105 copies/ml. For

individual countries on average, lowest genome loads were detected in Poland (1.9 ×

104 copies/ml) and highest in Italy (7.1 × 105 copies/ml). All 800 wild boar samples were

negative for PPgV RNA.

Table 2-1. Porcine pegivirus genome detection rates and viral genome load in serum

samples from individual animals and herds from different countries in Europe and

Asia 1. 1 PPgV, porcine pegivirus; pos., positive.

Table 2-2. Number of pegivirus positive pigs of different age groups from Europe and

China.

Chapter 2

20

Phylogenetic analyses. Altogether 31 PPgV partial NS3 sequences were obtained from

domestic pigs, of which nine were identical to one or more other sequences. In total,

ten sequences from Germany, three sequences from Italy, four sequences from Poland,

nine sequences from Great Britain and five sequences from China were acquired.

Sequences GER/SA/13, GER/SA/91, and PL/159 were identical to one additional

sequence each, while IT/77, GB/16, and GB/23 were identical to two further sequences

each. In Germany, Poland, and Italy, all identical sequences originated from samples

from the same farms, while herd affiliation was unknown for samples from Great

Britain.

Twenty-two distinct sequences shown here (Figure 2-1) were submitted to GenBank.

They displayed nucleotide sequence identities of >90%. According to phylogenetic

analysis, PPgV formed a separate branch in the tree of pegiviruses and viral sequences

segregated into two main clusters, one of which contained only sequences from

Europe (Germany, Great Britain and Poland). Within the second main cluster, some

branches contained sequences recovered from animals in Europe (GER/NDS/T72 and

IT/77) in close proximity to variants from China (i.e. CN/6/5) and USA (i.e.

33/ND/2017)24.

Overall, the most closely related pegivirus sequence found in other species when

compared to PPgV was bat pegivirus sequence PDB-1715 (GenBank KC796088), which

had a nucleotide sequence identity of 58.1% with PPgV/GB/30. A human pegivirus

type 2 sequence, ABT0070P.US (GenBank KT427411), had the lowest nucleotide

identity (47.1%) compared to PPgV sequences. When comparing PPgV sequences with

pegivirus sequences originating from horses, nucleotide identities ranged from 53.7%

to 55.7%. The sequence identities between PPgV and rodent pegivirus were around

54.0%, while the identities with simian pegiviruses ranged from 50.0% to 55.8%.

Chapter 2

21

Figure 2-1. Phylogenetic analysis of porcine pegiviruses from different countries and

other mammalian pegiviruses. Numbers along branches represent percentage

bootstrap values (bootstrap values < 80 % are not given). GenBank accession numbers

are in parentheses. Scale bar indicates nucleotide substitutions per site. PPgV

sequences are marked with a circle and the circle color indicates the country of origin.

Pegivirus species A-K are indicated on the right.

Chapter 2

22

PPgV RNA in tissue samples. In tissue samples of PPgV positive pigs, PPgV RNA

was most abundant in the liver (Table 2-3). Liver samples of animals A and B contained

343.9 and 142.5 viral RNA copies/mg tissue, respectively, while 119.3 copies/mg tissue

were found in the liver of animal C using qRT-PCR. Serum samples of these animals

contained 2,051.1 copies/µl (animal A), 388.6 copies/µl (animal B) and 157.0 copies/µl

(animal C). PBMCs were only available from animal A and contained 46 copies/µl

whole blood used for isolation (Table 2-3).

Table 2-3. Porcine pegivirus RNA quantities and fluorescence in situ hybridization

results in blood and different tissues from two domestic pigs from Germany 1. 1 PPgV,

porcine pegivirus; FISH, fluorescence in situ hybridization; GE, genome equivalents;

n.d., not determined; boldface indicates positive FISH results; *fresh blood was not

available.

Chapter 2

23

FISH was used to investigate the liver, thymus, PBMCs and different lymph nodes of

animal A, as well as the liver and thymus of animal B, and respective tissues of

negative control pigs. PPgV specific signals were detected in the liver of both PPgV

positive pigs (Table 2-3; Figure 2-2). Furthermore, several cells of the medullary and

cortical region of the thymus of animals A and B were observed to be virus positive

using the PPgV specific probe. Additionally, PBMCs of animal A were found to be

virus positive in FISH, while lymph nodes, spleen, tonsils, bone marrow and pancreas

of animal A tested virus negative. The non-probe incubation as well as the PPgV PCR-

negative pigs showed no detectable positive area in the same tested organs,

respectively. During necropsy of animal A, multifocal, mild, subendocardial

hemorrhages were present. Histopathology showed a mild, portally accentuated,

lymphohistiocytic hepatitis, a mild, diffuse infiltration of eosinophils within the

thymus, tonsils and lymph nodes and single multinucleated giant cells within the

medullary part of the thymus. Furthermore, lymph nodes revealed a mild, diffuse

sinus histiocytosis. A moderate, focal, perivascular, lymphoplasmahistiocytic,

partially eosinophilic dermatitis was present at the pinna. Additionally, a mild

endocardiosis, a mild, lymphohistiocytic epicarditis and a mild to moderate, focal,

follicular, lymphocytic conjunctivitis were observed.

Chapter 2

24

Figure 2-2. Fluorescence in situ hybridization of porcine pegivirus (PPgV) positive and

negative pigs using a PPgV specific probe; overlay phase contrast and

immunofluorescence; bar = 100 µm. (A) Single hepatocytes of the liver of a PPgV

positive pig showed an intracytoplasmic positive signal for PPgV using a PPgV

specific probe, also shown at higher magnification in the insert; arrows: nuclei of

hepatocytes surrounded by intracytoplasmic, red, positive signals. (B) The liver of a

PPgV negative pig lacked a PPgV specific signal. (C) Within the thymus of a PPgV

positive pig, scattered cells showed an intracytoplasmic red positive signal for PPgV,

also shown at higher magnification in the insert. (D) Within the thymus of a PPgV

negative pig, all cells were negative for PPgV using a PPgV specific probe. (E) Several

PBMCs from a PPgV positive pig showed a red positive signal using a PPgV specific

probe, also shown at higher magnification in the insert. (F) PBMCs from a PPgV

negative pig were negative for PPgV.

Chapter 2

25

Discussion

The genus Pegivirus has grown in recent years, as new viruses were identified in

different hosts. Yet little is known about their pathogenicity and the impact on the

host’s immune response. In this study, our aim was to gain detailed insights into the

distribution of PPgV in different parts of the world and the genetic diversity of PPgV.

Viral RNA was detected in serum samples from domestic pigs from various European

countries and China, with an overall individual detection rate of 2.7%.

Investigation of three different age groups from Europe and Asia showed a lower

PPgV positive rate in younger animals such as piglets (1.9%) and fattening pigs (1.2%)

than in adult animals (3.4%). This observation is concordant with the results from a

recently published study from China; however, the increase in PPgV positive rate was

more prominent there (1.6–11.3%). Focusing on samples from China, we found similar

results: 1.0% detection rate in piglets and 9.7% detection rate in sows and boar25.

The PPgV positive rates found in this study differ between countries. While no

samples were PPgV positive from Switzerland, Serbia and Taiwan, samples from

Germany, Poland and Italy have a positive rate similar to the one described previously

for German domestic pigs (2.2%)9. High detection rates in China (7.8%) and Great

Britain (10.3%) found here are nonetheless lower than the positive rate observed in the

USA (15.1%) in a previous study24. In humans, HPgV prevalence ranges from 0.5 to 5%

in healthy blood donors from developed countries, but is higher in blood donors from

developing countries (5–18.9%), and in individuals co-infected with blood borne or

sexually transmitted diseases, like hepatits C virus or HIV-130–32. Equine pegivirus

(EPgV) has been found in 12 of 328 horses (3.7%) from Europe and 7 of 74 horses (9.5%)

from USA4,12, thus showing similar detection rates as PPgV. The divergence in PPgV

detection rates suggests uneven distribution of virus infection and local spread of

PPgV. This may be caused by the occurrence of other infectious diseases in pig

Chapter 2

26

populations, similar to observations in humans with co-infection, and needs to be

studied further. The viral loads determined here (2.4 × 102 to 2 × 106 copies/ml) are

similar to EPgV RNA loads described in one study, which ranged from 3.2 × 104 to 3.2

× 106 RNA copies/ml4. However, another study found higher EPgV viral loads (4.1 ×

105 to 2.0 × 109 RNA copies/ml)12, and the mean RNA load of HPgV in human plasma

typically reaches >1 × 107 copies/ml. This may suggest lower replication of PPgV in

vivo compared to HPgV and EPgV.

Although HPgV does not appear to be hepatotropic, high amounts of PPgV RNA in

the porcine liver shown by qRT-PCR and in situ techniques suggest that viral RNA

may accumulate in the liver or even that PPgV has the ability to replicate in

hepatocytes. However, this hypothesis will have to be investigated in future studies,

as well as whether PPgV infections might be the cause of histopathological changes in

the liver, as seen in animal A. Moreover, presence of PPgV RNA in PBMCs and in the

thymus supports lymphotropism analogical to HPgV22. Positive FISH results in

primary but not secondary lymphoid organs, such as spleen or lymph nodes, imply

that the virus might replicate in the thymus and spread to other tissues (e.g. the liver)

via PBMCs, but successfully evades recognition by the immune system, which could

lead to a persistent infection in the host. Despite significant amounts of viral RNA

detected in cells and tissues, highest viral loads were present in the serum of infected

animals. With regard to this, low amounts of PPgV RNA in further organs and tissues

can most probably be attributed to blood residues. Possible presence of viral RNA in

serum microvesicles and associated virus uptake by PBMCs, as seen for HPgV, remain

to be determined22.

Phylogenetic analyses showed close genetic relationships among PPgV sequences

from different countries, like sequences GER/NDS/T72 and CN/6/5. This could suggest

virus spread by international trade with pigs or pig products, such as feed. While all

wild boar samples were tested negative for PPgV RNA in this study, other porcine

Chapter 2

27

viruses from the family Flaviviridae, such as APPV, have been shown to be present at

a higher rate in wild boar (19%) than in domestic pigs from Germany (6.2%)29,33. For

APPV, virus transmission between wild boar and domestic pigs appears likely, as

strains originating from wild and domestic animals show genetic distance of as little

as 6.6%29. However, due to the comparatively low prevalence of PPgV in Germany,

transmission of the virus from domestic pigs to wild boar and vice versa may be

limited. Only samples from wild boar hunted in northern Germany entered the present

study. Future studies with extended sampling will reveal whether PPgV is also absent

in wild boar from other geographical regions. As genome detection alone may result

in underestimation of virus dissemination, upcoming investigations of samples from

domestic pigs, wild boar and other species will also address serological reactions upon

infection with PPgV.

These results manifest that PPgV, like other pegiviruses, is distributed over several

continents. It can be hypothesized that putative immune modulatory effects of PPgV

infections are implicated in pig health worldwide. Detection of PPgV RNA in

lymphoid cells suggests that the virus has the potential to affect the immune system of

pigs. First insights into the cell- and organ tropism of PPgV suggest that the virus may

be hepatotropic and/or lymphotropic. Future studies will clarify the pathogenic

potential and immune modulatory effects of this newly discovered, widely distributed

virus.

Methods

1,736 serum samples from domestic pigs from different countries in Europe (Germany,

Great Britain, Poland, Switzerland, Italy and Serbia) and Asia (mainland China and

Taiwan) originating from 132 different herds were analyzed in this study. For samples

collected in Great Britain, the number of herds was unknown. Samples included 108

piglets up to four weeks old, 923 fattening pigs over four weeks old, 557 sows and

Chapter 2

28

boar, and 148 pigs of unknown age and use. Samples were taken between 2014 and

2018, originated from apparently healthy domestic pigs and were taken within the

framework of national veterinary health management in concordance with national

legal and ethical regulations. Residual volumes of these samples were provided for

use in the current study, therefore no ethical approval was required for use of these

samples. In addition, 800 serum samples from hunted wild boar from Lower Saxony,

Germany, were included. 456 of these wild boar samples were collected during the

hunting seasons of 2015/2016 and 2016/2017 and were used in a previous study

investigating APPV prevalence29. 344 additional wild boar samples were collected

during the hunting season of 2017/2018. Furthermore, blood and post-mortem tissue

samples originated from apparently healthy PPgV positive pigs (n = 3, animals A, B,

and C) from the Clinic for Swine, Small Ruminants, Forensic Medicine and

Ambulatory Services (University of Veterinary Medicine, Hannover) and PPgV

negative control pigs (n = 2). To rule out presence of co-infections with APPV and

porcine reproductive and respiratory syndrome virus (PRRSV), PPgV positive animals

were also tested using RT-PCR and found negative for both viruses (data not shown).

One pig (animal A) was submitted to the Department of Pathology, University of

Veterinary Medicine Hannover. A full necropsy was performed and samples of 40

different tissues were collected and stored at −80 °C or fixed in 10% neutral buffered

formalin and embedded in paraffin wax. For histopathological examination, 3 µm

thick sections were stained with hematoxylin and eosin. Different organ and tissue

samples and a liver sample originated from two further PPgV positive pigs, animal B

and animal C, respectively. Control samples for FISH were taken from PPgV negative

pigs. PBMCs from animal A and one negative control animal were isolated from ~1 ml

blood by density gradient centrifugation with Histopaque (Merck, Darmstadt,

Germany). Euthanasia and sampling were approved by Lower Saxony’s official

authorities (LAVES AZ 15A602 and 17A195) and were carried out in accordance with

German legislation (TierSchVersV).

Chapter 2

29

RNA was isolated from 140 µl of serum using the QIAmp Viral RNA Mini Kit

(QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Isolation

of RNA from preweighed tissue samples was achieved using the NucleoSpin RNA kit

(Macherey-Nagel, Düren, Germany) or the RNeasy Mini Kit (QIAGEN) according to

the manufacturer’s instructions and RNA samples were stored at −80 °C until testing.

For PPgV genome quantification, a TaqMan based qRT-PCR targeting the highly

conserved NS3 encoding region with primers PPgV/ fwd/7

(5′-GTCTATGCTGGTCACGGA-3′), PPgV/rev/8

(5′-CACTCATCGCAAATGACCAC-3′) and probe PPgV/pro/11

(5′-[6FAM]-CCATTTCGCGAACCACTGATTCCA-[BHQ1]-3′) was developed and

verified using samples that were PPgV positive in a SYBR Green qRT-PCR (QIAGEN)

described in an earlier study9. For the new PCR assay, an in vitro transcribed RNA copy

standard was developed using MEGAscript Kit (ThermoFisher Scientific, Germany) to

allow for absolute quantification of genome copies. Real-time RT-PCR was performed

using the Mx3005P QPCR System (Agilent Technologies, Santa Clara, USA) and the

QuantiTect Probe RT-PCR Kit (QIAGEN) according to the manufacturer’s instructions

on samples and RNA standard dilutions. Briefly, 12.5 µl RT-PCR master mix, 0.25 µl

reverse transcriptase, 0.8 pmol of each primer and 0.2 pmol of the probe, 5.25 µl water

and 5 µl sample RNA were used in each reaction of 25 µl with the following

temperature profile: 50 °C for 30 minutes, 95 °C for 15 minutes and 40 cycles of 94 °C

for 15 seconds and 60 °C for 1 minute. Serum samples were initially screened in pools

containing three to ten individual samples; subsequently samples from positive pools

were tested individually.

For phylogenetic analysis, amplicons corresponding to a partial NS3 coding sequence

were generated by one of the following two methods: a) RT-PCR with SuperScript III

One-Step RT-PCR System with Platinum TaqDNA Polymerase (Life technologies,

Germany) with primers PPgV/fwd/G1 (5′-CACCGGGCTGTTTCTGCTA-3′) and

PPgV/rev/G4 (5′-TTCCTTCCACACCAACCCAT-3′), or b) cDNA synthesis with

Chapter 2

30

SuperScript II Reverse Transcriptase (Invitrogen, Germany) using random hexamers

followed by nested PCR with outer primers PPgV/fwd/G1 and PPgV/rev/G4, and inner

primers PPgV/fwd/G3 (5′-CGGGCTGTTTCTGCTAGGT-3′) and PPgV/rev/G2

(5′-CACCAACCCATCGAGGATCA-3′) using Taq polymerase included in the

Maxima Hot Start Green PCR Master Mix (2X) (ThermoFisher Scientific) and the

following cycling parameters: 95 °C for 4 min, 40 cycles of 95 °C for 30 s, 52 °C for 30

s, 72 °C for 75 s, and 72 °C for 10 min. PCR products with an expected length of 1,290

(method a) and 1,278 (method b) were purified using the GeneJET PCR Purification

Kit (ThermoFisher Scientific) according to the manufacturer’s instructions and

submitted to Sanger sequencing (FlexiRun, LGC Genomics, Germany) with primers

PPgV/fwd/G3 and PPgV/rev/G2. Sequences were trimmed to a final length of 1041

base pairs and a multiple sequence alignment was performed with ClustalW

implemented in BioEdit 7.034. Phylogenetic trees were calculated in MEGA7 using the

Maximum-likelihood method and the Kimura 2-parameter substitution model35 with

500 replicates for statistical evaluation.

FISH was performed on formalin-fixed, paraffin-embedded organ sections of two qRT-

PCR positive pigs (animal A and B) and on the PBMC pellet of one pig (animal A)

using a PPgV specific RNA probe covering parts of the PPgV NS3. The probe set

(ViewRNA TYPE 1 Probe Set, ThermoFisher Scientific) covered positions 2–816 of a

target sequence with 1,172 nucleotides that overlapped with the partial PPgV sequence

of animal A (GenBank MH979651). The procedure was carried out according to the

manufacturer´s protocol with minor variations as previously described (ViewRNA

TYPE 1 Probe Set; ViewRNA™ ISH Tissue Assay Kit (1-plex) and ViewRNA

Chromogenic Signal Amplification Kit; ThermoFisher Scientific;)36. Briefly, sections

were deparaffinized, boiled in pretreatment solution® at 90 °C for 10 minutes, digested

by a protease QF® at 40 °C for 10 minutes and afterwards fixed. Hybridization to the

specific probe was performed for 6 hours. Following preamplification and

amplification steps, sections were stained with Fast Red Substrate and counterstained

Chapter 2

31

with Mayer´s hemalum® (Carl Roth GmbH, Karlsruhe, Germany). Images were

acquired with an inverted fluorescence microscope (Olympus IX-70; Olympus Life

Science Europe GmbH, Hamburg, Deutschland). The specificity of the probe was

confirmed by including a non-probe incubation which served as system negative

control and organ sections and cells of PPgV RT-PCR-negative pigs, respectively.

Accession codes. The obtained DNA sequences were deposited in GenBank (accession

numbers: MH979651-MH979672).

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Acknowledgements

We thank Vesna Milićević (Belgrade, Serbia), Ming-Chung Deng and Chia-Yi Chang

(New Taipei City, Taiwan), Serum Bank of the Federal Food Safety and Veterinary

Office (Bern, Switzerland), Animal and Plant Health Agency (Weybridge, UK),

Francesco Feliziani and Gian Mario De Mia (Perugia, Italy), Katarzyna Podgórska and

Katarzyna Stępniewska (Puławy, Poland), Hua-Ji Qiu and Yuan Sun (Harbin, China),

Sarah Derking and Jörg Tenhündfeld (Vreden, Germany) and Thomas Große Beilage

(Essen/Oldenburg, Germany) for providing porcine serum samples. We highly

appreciate the help of Inga Grotha concerning sample database set-up and RNA

preparation. This study was supported by the German Center for Infection Research

(DZIF), Thematic Translational Unit “Emerging Infections”, grant number 8002801801

as well as by the European Union’s Horizon 2020 research and innovation program

under grant agreement No. 643476 (COMPARE) and the Deutsche

Forschungsgemeinschaft (DFG, German Research Foundation) – 398066876/GRK

2485/1. This publication was further supported by the Deutsche

Forschungsgemeinschaft and University of Veterinary Medicine Hannover,

Foundation within the funding programme “Open Access Publishing”.

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35

3 Dissecting antibody reactivity and possible transmission routes in

porcine pegivirus infection

Kennedy J, Hoeltig D, Becher P, Baechlein C.

This chapter is a manuscript in preparation.

Contribution as first author:

Experimental work: Sample preparation of serum and excretion samples, RNA

isolation, screening for PPgV RNA presence by TM qRT-PCR, RT-PCR and sequencing

of PPgV in serum samples, cloning of plasmids and bacterial expression of PPgV

proteins, establishment of purification protocol for PPgV NS3h protein, establishment

of SDS-PAGE and Western blot protocols for testing serum sample reactivity with

PPgV proteins, screening of serum samples for PPgV-specific antibodies by ELISA.

Evaluation and scientific presentation of the results: Analyses and graphical

presentation of qRT-PCR results, presentation of Western blot and antibody-ELISA

results. Scientific writing: preparation of the manuscript, tables and figures.

Chapter 3

36

Abstract

Porcine pegivirus (PPgV) is a positive-sense ssRNA virus belonging to the Pegivirus

genus within the Flaviviridae family that can cause persistent infections in its host. The

association of PPgV infection with disease remains unknown, but viral genome has

been detected in up to 15.1% of pigs from the United States, China and Europe. Thus

far, there are no reports on the virus transmission routes and assays for the detection

of PPgV-specific antibodies (Abs) are not available. Therefore, this study investigated

serum and excretion samples from PPgV viremic pigs to determine possible virus

shedding. Additionally, we expressed PPgV non-structural protein 3 helicase domain

(NS3h) and C-terminally truncated E2 (E2t) intracellularly in E. coli for use in Ab

detection. Purified NS3h showed reactivity with porcine serum in Western blot (WB)

and indirect enzyme-linked immunosorbent assay (ELISA), and crude E2t (from

bacterial inclusion bodies) was reactive in WB. Thus both, NS3h and E2t, appear to be

immunogenic and are viable candidates for the further validation of ELISA methods.

Detection of PPgV RNA in serum and excretion samples of PPgV viremic pigs and

their piglets revealed one piglet born of a sow, which cleared viremia by day 69 of

gestation, was positive in serum directly after birth (before colostrum intake),

indicating intrauterine infection. Lack of PPgV genome detection in excretion samples

suggested that PPgV may rather be transmitted horizontally via the blood-borne route.

This study reports the first methodological steps towards investigating the immune

response induced by PPgV and provides insights into possible viral transmission

routes.

Chapter 3

37

Introduction

Porcine pegivirus (PPgV) was first discovered in 2016 in apparently healthy domestic

pigs from Germany (Baechlein et al., 2016). It belongs to the genus Pegivirus, which

comprises a group of positive-sense, single-stranded RNA viruses within the

Flaviviridae family (Smith et al., 2016). Persistent pegivirus infections have been found

in humans and horses and were also present in three pigs from Germany, in which

viral RNA was detected for up to 22 months (Berg et al., 1999, Tanaka et al., 1998,

Kapoor et al., 2013a, Baechlein et al., 2016). As in other pegiviruses, the pathogenicity

of PPgV is so far unknown. Although PPgV RNA has been found in porcine serum

samples from a farm with pigs exhibiting vesicular disease and lameness, there is no

clear association between PPgV infection and disease, and most frequently the virus

has been detected in apparently healthy pigs with no clinical signs of viral infection

(Yang et al., 2018, Baechlein et al., 2016, Kennedy et al., 2019).

PPgV is widely distributed and has been detected in domestic pigs from North

America, China, and different countries in Europe (Yang et al., 2018, Lei et al., 2019,

Chen et al., 2019, Baechlein et al., 2016, Kennedy et al., 2019). The highest RNA

detection rate was reported in 11 of 67 (16.4%) clinical serum or tissue samples from

China (Chen et al., 2019). However, reports on PPgV-specific antibody (Ab) detection

are lacking and viral genome detection alone may result in the underestimation of the

abundance of PPgV occurrence in domestic pig herds and further potential hosts.

Ab detection is an important diagnostic tool for HPgV, as viremia and envelope

protein 2 (E2) Ab response are mutually exclusive markers of infection in most

individuals (Stapleton et al., 2011). Because of this, investigations on the exposure rate

of HPgV require both E2-Ab and RNA detection (Gutierrez et al., 1997, Thomas et al.,

1998). Different serological assays have been established by expression of full-length

or C-terminally truncated E2 protein in E. coli or mammalian cells (Chinese hamster

Chapter 3

38

ovary (CHO) and Baby hamster kidney (BHK-21) cells) (Dawson et al., 1996, Dille et

al., 1997, Pilot-Matias et al., 1996a, Tacke et al., 1997a). In most developed countries,

HPgV RNA detection rates and HPgV E2-Ab-positive rates in volunteer blood donors

amount to 1-4% and 5-13%, respectively, with higher rates in developing countries

(Blair et al., 1998, Gutierrez et al., 1997, Pilot-Matias et al., 1996a, Tacke et al., 1997b,

Mohr and Stapleton, 2009, Williams et al., 2004). HPgV RNA and Ab are more

prevalent in high risk groups, such as intravenous drug users or people suffering from

other blood-borne or sexually transmitted infections, reaching exposure rates over 80%

(Williams et al., 2004, Scallan et al., 1998, Tacke et al., 1997b, Stapleton et al., 2011).

Clearance of HPgV from blood occurs within two years in most immunocompetent

individuals and coincides with the development of protein conformation-dependent,

long-lasting anti-E2 Abs (Berg et al., 1999, Tanaka et al., 1998, McLinden et al., 2006,

Pilot-Matias et al., 1996b, Tacke et al., 1997b). Due to the fact that the Ab response

appears to be restricted to E2, this antigen is assumed to possess the immunodominant

epitopes (McLinden et al., 2006).

Equine pegivirus (EPgV) RNA has been detected in 0.8% to 14.2% of horses from the

United States, China and Brazil (Kapoor et al., 2013a, Lyons et al., 2014, Lu et al., 2016,

Agnello et al., 1999, Figueiredo et al., 2019). An ELISA for the detection of Abs against

this virus was established using the non-structural protein 3 helicase domain (NS3h)

and bacterial expression in E. coli (Lyons et al., 2014). In the study, 218 of 328 (66.5%)

horses were positive for NS3h Abs in ELISA, of which 88% (192 of 218) were confirmed

by Western blot (WB). Contrary to findings in HPgV with E2 Ab assays, in the NS3 Ab

assay for EPgV, 10 of the 12 RT-PCR positive horses were also Ab positive (Lyons et

al., 2014).

Transmission of human pegivirus can occur both parenterally through blood and

sexual contact, as well as vertically (Dawson et al., 1996, Linnen et al., 1996, Schmidt et

al., 1996, Feucht et al., 1996). The transmission of EPgV has not been studied in detail.

Chapter 3

39

However, transmission of Theiler’s disease associated virus (TDAV, Pegivirus E)

between horses has been shown to be possible by experimental inoculation

(Chandriani et al., 2013). Similarly, pegiviruses can be transmitted to different species

of New World monkeys by experimental infection via the blood-borne route (Stapleton

et al., 2011). Transmission routes of pegiviruses infecting bats were not extensively

studied; however, viral RNA was detected in saliva of viremic bats, suggesting that

horizontal or even zoonotic transmission may be possible (Epstein et al., 2010).

To our knowledge, an assay for the detection of PPgV-specific Abs has not been

described and studies on virus transmission between pigs have not been reported.

Therefore, we expressed PPgV proteins NS3h and C-terminally truncated E2 (E2t)

intracellularly in E. coli and implemented WB and indirect ELISA methods for the

detection of PPgV-specific serum Abs. A serological assay will permit the investigation

of PPgV infection characteristics, including time point of seroconversion, relevance

and relation of IgM, IgG, and IgA Abs, as well as maternally derived Abs. During the

study, PPgV field infected domestic pigs were monitored alongside their piglets and

serum and excretion samples were obtained every 2-3 weeks for evaluation of PPgV

RNA presence and potential serological responses. Taken together, this study provides

the first insights into possible PPgV transmission routes and PPgV-specific Ab

detection.

Materials and methods

Samples

Serum samples available for Ab detection originated from apparently healthy

domestic pigs from Europe (Germany, Italy, Poland, Great Britain, Switzerland and

Serbia) and Taiwan, which were used in a previous study on PPgV RNA detection rate

(Kennedy et al., 2019). They were collected between 2014 and 2018 within the

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40

framework of national veterinary health management in concordance with national

legal and ethical regulations.

Additionally, serum as well as nasal swabs, oral swabs, and fecal and urine samples

were collected from six healthy, originally PPgV positive pigs (animals A-F) from a

farm in Lower Saxony, Germany, and from initially PPgV negative control pigs

(animals G, H, J, and K) from the Farm for Education and Research in Ruthe of the

University of Veterinary Medicine Hannover. All animals were housed in the Clinic

for Swine and Small Ruminants and Forensic Medicine and Ambulatory Service of the

University of Veterinary Medicine Hannover after the initial PPgV RNA screening and

were sampled every 2-3 weeks for a maximum of 46 weeks (see Figure 3-4). Excretion

samples were taken from all animals at each sampling time point from week 7 to week

46.

Animals A and B, both sows, were inseminated in week 20 while both animals were

PPgV RNA positive. All 13 piglets from sow A (P1-P13) and 13 piglets from sow B

(P14-P26) were sampled after birth (before colostrum intake), and every 2 weeks

thereafter for a maximum of 12 weeks. Samples taken from piglets included serum,

nasal, oral and fecal swabs. For the detection of PPgV RNA in piglet excretion samples,

swabs of three piglets of each sow were selected for each sampling week. Two vaginal

swab samples were taken from each sow during the birth of their piglets. Sampling

was approved by Lower Saxony’s official authorities (LAVES AZ 15A602) and was

carried out in accordance with German legislation (TierSchVersV).

All swab samples were soaked in 1 ml cell culture media containing antibiotics for a

minimum of 2 hours (h), after which media was used for RNA isolation, or stored.

Feces were diluted 1:10 in phosphate buffered saline (PBS), vortexed vigorously and

centrifuged for 20 min at 4000 × g, and supernatant was stored. All samples were

stored at -80 °C until use.

Chapter 3

41

RNA isolation and PCR

Viral RNA was isolated from 200 µl of serum (100 µl of serum from piglets in week 36

due to limited volume available), urine, fecal swab supernatant, or media using the

IndiMag Pathogen Kit (Indical Bioscience, Germany) and the KingFisher Duo Prime

extractor (Thermo Scientific, Germany) and was eluted in 100 µl elution buffer.

Quantitative reverse-transcription PCR (qRT-PCR) was used to detect PPgV RNA as

previously described (Kennedy et al., 2019). Control RNA was added to excretion

samples and detected by qRT-PCR (primers EGFP-1-F

(5’-GACCACTACCAGCAGAACAC-3’), and EGFP-2-R

(5’-GAACTCCAGCAGGACCATG-3’) and probe EGFP-HEX

(5’-[HEX]-AGCACCCAGTCCGCCCTGAGCA-[BHQ1]-3’)) to confirm successful

RNA isolation, as previously described (data not shown) (Hoffmann et al., 2006).

Complementary DNA of the viral genomic RNA of PPgV isolate PPgV_903/Ger/2013

(GenBank accession number KU351669.1) was synthesized using SuperScript II

Reverse Transcriptase (Invitrogen, Germany). PCR for the amplification of the

genomic regions encoding the predicted NS3 helicase domain and the truncated E2

was performed using primers PPgV/NS3H/fwd/19

(5’-ATTACTCGAGGTGGTCCCCTGGGCCAACATGCCTCAGGA-3’) and

PPgV/NS3H/rev/20

(5’-CCGCAGATCTGTCATACCACAATCAGTCACAGTGTCA-3’), or

PPgV/E2T/fwd/17 (5’-ATTACTCGAGCTTCTGCTCCTTGCTGCTGCTG-3’) and

PPgV/E2T/rev/18 (5’-CCGCAGATCTGTAGGAAACTGGTCTGTGTACTCAT-3’),

respectively, containing appropriate restriction sites for subcloning (XhoI and BglII,

underlined).

Chapter 3

42

Expression of PPgV NS3 helicase and truncated E2 recombinant proteins

The NS3h (811 bp) and E2t (994 bp) amplicons were cloned into a pET-19B vector

(Novagen, Germany) downstream of a polyhistidin tag. Plasmid and inserts were

digested using appropriate restriction enzymes (Thermo Scientific) and were purified

using the GeneJet Gel Extraction kit (Thermo Scientific) followed by ligation with T4

ligase (New England Biolabs, Germany). Recombinant E. coli Top10 clones were

grown in LB-medium and were selected with ampicillin (50 µg/ml). Plasmid integrity

was confirmed by PCR and sequencing.

For protein expression, One Shot BL21(DE3)pLysS chemically competent E. coli

(Invitrogen) were used according to the manufacturer’s instructions. Fresh cultures

were added to 200 ml LB-medium containing 50 µg/ml ampicillin and cultured at 37

°C and 250 rpm. At an optical density at 600 nm of 0.6, bacterial expression was

induced with 1 mM Isopropyl-beta-D-thiogalactopyranosid (IPTG) for 2 h. Bacteria

were centrifuged at 4000 × g for 20 min at 4 °C. Cell pellets were weighed and frozen

at 20 °C. B-PER complete protein extraction reagent (Thermo Scientific) was used for

lysis of bacteria and soluble and insoluble cytoplasmic fractions were analyzed for the

presence of NS3h and E2t proteins in Coomassie blue stained sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) gels and Western blot. For E2t

protein, the insoluble cytoplasmic fraction was washed four times with B-PER diluted

1:10 and the remaining pellet was resuspended in 1% SDS for use in Western blot.

Purification of PPgV NS3 helicase recombinant protein

Soluble proteins containing NS3h were diluted 1:10 in fast protein liquid

chromatography (FPLC) buffer (20 mM sodiumhydrogenphosphate, 500 mM

sodiumchloride, pH 7.4) and imidazole was added to a final concentration of 40 mM.

The diluted sample was filtered through a 0.22 µm filter membrane. Protein was

purified using the Äkta Pure FPLC and a HisTrap excel 1 ml column (GE Healthcare,

Chapter 3

43

Sweden). After sample application (flow rate 1 ml / min), the column was washed with

20 ml FPLC buffer containing 40 mM imidazole and protein was eluted in 10 fractions

of 0.5 ml each of FPLC buffer containing 500 mM imidazole. Elution fractions were

analyzed by SDS-PAGE and Coomassie blue staining, as well as WB. Protein

concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo

Scientific). Fractions containing high concentrations of target protein were pooled and

stored at 4 °C containing 0.1% sodium acid as preservative.

SDS-PAGE, Coomassie blue staining and Western blot

To visualize bacterial protein expression and to confirm FPLC-purification of NS3h

protein, diluted bacterial lysate containing crude NS3h or E2t protein, as well as eluted

fractions of NS3h, were analyzed on 12.5% SDS-PAGE gels stained with Coomassie

blue or blotted onto PVDF membranes in a semidry electroblotting procedure.

Membrane blocking, Ab incubation and washing steps were carried out with TBS-0.1%

Tween20-buffer. PVDF membranes were blocked overnight with 2% Amersham ECL

Prime Blocking Reagent (GE Healthcare). Membranes were then incubated with Anti-

His6 mouse monoclonal Ab (Roche, Germany) diluted 1:100 for 1 h at RT and then

washed four times for 15 min. Polyclonal Rabbit Anti-Mouse Immunoglobulins / HRP

(Dako, Denmark) diluted 1:2,000 was then added and incubated again for 1 h at RT,

followed by four more steps of washing. Amersham ECL Prime Western Blotting

Detection Reagent (GE Healthcare) was used for the visualization of bound Ab.

Serum samples were tested on WB membranes obtained after SDS-PAGE and transfer

of purified NS3h or crude E2t was performed as described above. Membranes were

blocked as described above and incubated with serum diluted 1:500 in blocking

reagent for 1 h on a shaker at RT. Membranes were washed and anti-pig IgG (whole

molecule)-Peroxidase antibody produced in rabbit (Sigma Aldrich, Germany) diluted

1:30,000 was added for 1 h at RT, followed by another round of washes. Bound Ab was

Chapter 3

44

detected using ECL Prime or ECL Select Western Blotting Detection Reagents (GE

Healthcare).

ELISA

Nunc Medisorp ELISA plates (Thermo Scientific) were coated with 250 ng / well

(ELISA250) or 100 ng / well (ELISA100) of NS3h in 100 µl coating buffer (0.1 M NaCO3,

pH 9.6) per well over night at 4 °C. Plates were washed three times with 360 µl PBS-

0.05% Tween20 (PBS-Tw) and blocked with 4% skimmed milk powder in PBS-Tw for

2 h at room temperature (RT) and subjected to another round of washes. Porcine serum

samples were diluted 1:25 in 4% skimmed milk powder in PBS-Tw and 100 µl per well

were incubated for 1 h at 37 °C. Plates were washed again and anti-pig IgG (whole

molecule)-Peroxidase produced in rabbit (anti-pig IgG, Sigma Aldrich) diluted

1:35,000 in 4% skimmed milk powder in PBSM-Tw was added and incubated for 1 h

at 37 °C. After washing, tetramethylbenzidine (Sigma Aldrich) was added and plates

were incubated in the dark for 10 min at RT. The reaction was stopped with 1 M

hydrochloric acid and optical densities (ODs) were determined automatically (TECAN

Sunrise Remote, Tecan, Switzerland) at a wavelength of 450 nm and a reference

wavelength of 620 nm.

Results

NS3h protein purification

Purification of NS3h was achieved by immobilized metal ion chromatography (IMAC)

in FPLC, which permitted elution of 0.67 mg protein / ml. Western blots and

Coomassie blue stained gels were used to assess the elution and the purity of the target

protein. Though weak bands of unspecific protein were visible at larger protein sizes,

purity of NS3h was high (Figure 3-1).

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45

Figure 3-1. Coomassie gel of NS3h protein before and after purification by IMAC. Lane

1 shows crude soluble protein that was applied onto the HisTrap column before

dilution with FPLC buffer. Lane 2 shows flow through of FPLC, indicating proteins

that did not bind to the column. Lanes 3, 4 and 5 show different fractions of eluted

proteins with NS3h at a size around 37 kDa (compare Figure 3-2, lane 1: detection of

NS3h by the anti-His6 Ab. Additional unspecific bands can be seen in lanes 3 and 4 at

a protein size of ~70 kDa.

NS3h-specific serum antibody reactivity in Western blot and ELISA

PPgV RNA positive and negative serum samples were prescreened using ELISA250 to

identify samples suitable for validation of WB and ELISA (data not shown). Further

evaluation of 18 samples with varying reactivity in the preliminary ELISA250 was

performed by WB and ELISA100. Clearly visible NS3h-specific bands at a protein size

of ~37 kDa were seen following incubation with 5 of 18 samples (Figure 3-2). One

sample evidenced unspecific bands at a protein size ~70 kDa, and variations in

unspecific background reactions were visible, making evaluation of possible bands

difficult in 6 samples. Differences between ODs of ELISA250 and ELISA100 ranged from

a factor change of 0.6 (sample in lane 12, Figure 3-2) to 0.92 (sample in lane 3, Figure

3-2; Table 3-1).

Chapter 3

46

Figure 3-2. Western blots of purified NS3h protein incubated with serum samples as

first antibody (Ab). Lane 1 shows the position of the NS3h-His fusion protein at a

molecular weight of ~37 kDa using the anti-His6 Ab. Lanes 8, 11, 14, 17, and 18 evidence

an NS3h-specific band. Background varies from low (lanes 7, 9, 13, 14, 15, and 19) to

high (lanes 4, 5, and 18). Unspecific bands at a protein size ~70 kDa can be seen in lane

6.

Chapter 3

47

Lane no.

in NS3h

WB

ELISA250

OD

ELISA100

OD

WB

NS3h

band

WB

unspecific

bands

WB

background

PPgV

RNA

17 1.91 1.5 + + -

6 1.61 1.18 (+) + + -

14 1.58 1.19 + - +

18 1.57 1.28 + ++ +

11 1.45 1.08 + + -

4 1.08 0.84 (+) ++ -

2 1.04 0.8 (+) + +

8 0.93 0.61 + + +

3 0.82 0.75 (+) + +

5 0.82 0.64 (+) ++ -

19 0.76 0.52 - -

10 0.67 0.45 (+) + -

12 0.64 0.44 + -

15 0.38 0.23 - -

9 0.13 0.09 - +

16 0.11 0.1 + -

7 0.09 0.07 - +

13 0.07 0.04 - -

Table 3-1. Characterization of selected serum samples. ELISA optical densities (OD)

of serum samples and their reactivity with NS3h and background Western blot, as well

as PPgV RNA detection. Samples are arranged in descending order of their OD in

ELISA250 (arrow). Detection of NS3h in WB is indicated as positive [+], uncertain [(+)],

or negative [-].

E2t-specific serum antibody reactivity in Western blot

The expression of C-terminally truncated E2 was successfully achieved in the E. coli

strain BL21(DE3)pLysS used here, and the protein was detectable in the insoluble

cytoplasmic fraction of the bacterial lysate using the anti-His6 Ab in WB at a protein

size of ~40 kDa (Figure 3-3, lane 1). Due to detection of the target protein E2t in the

insoluble fraction indicative of expression in bacterial inclusion bodies, crude protein

containing E2t was prepared for WB analyses by repeated washing of the insoluble

protein pellet and final resuspension in 1% SDS.

Chapter 3

48

Two samples (one of which is seen in lane 17 of Figure 3-2; the other is not shown here)

that evidenced NS3h-specific Ab reactivity in WB and ELISA were implemented in

WB with E2t crude protein (equivalent to isolation from 100 µl of bacteria), one of

which evidenced an E2t-specific band (Figure 3-3, compare with lane 17 in Figure 3-2).

Figure 3-3. Western blot of crude E2t protein incubated with serum samples that

showed NS3h-specific antibody (Ab) reactivity in Western blot and ELISA as first Ab.

Lane 1 shows the position of the E2t-His fusion protein at a molecular weight of ~40

kDa using the anti-His6 Ab. An E2t-specific band is visible in lane 2, while unspecific

bands at a protein size ~35 kDa are visible in lanes 2 and 3.

PPgV RNA in serum and excretion samples of pigs and piglets

Six fattening pigs from a farm in Lower Saxony, Germany, were found to be PPgV

RNA positive in serum and were moved to the Clinic for Swine and Small Ruminants

and Forensic Medicine and Ambulatory Service of the University of Veterinary

Medicine Hannover. Animals A and B were continually tested PPgV genome positive

(samples taken every 2-3 weeks) by qRT-PCR for 38 and 27 weeks, respectively (Figure

3-4). Animals C, D, E, and F were PPgV RNA negative at the second sampling in week

3 and only animal D was again found PPgV RNA positive from weeks 13 to 33.

Additionally, excretion samples, including nasal and oral swabs, urine and feces, were

taken at each time point (from week 7 to week 46) to assess possible routes by which

Chapter 3

49

PPgV might be spread, all of which were found to be negative for PPgV RNA. Initially

PPgV genome negative control animal G was found to be PPgV RNA positive in serum

in week 17. Other negative control animals H, I and J were tested negative in serum

and excretion samples throughout the study.

Animals A and B were inseminated in week 20. While animal A remained PPgV RNA

positive for the duration of the pregnancy and until 12 days post-partum, animal B

was last found PPgV genome positive in week 27, 50 days after insemination. In week

36, both sows bore 13 piglets each. PPgV RNA was detected in serum of one piglet (P8)

directly after birth (before colostrum intake) in week 36 and contained 1,412 RNA

copies / ml serum (Cq value 37.61). All other piglet serum samples were tested PPgV

RNA negative by qRT-PCR. For each sampling week, excretion samples were selected

from three piglets of each sow for detection of PPgV genome, and samples from serum

RNA positive piglet P8 in week 36 were additionally included. However, all excretion

samples from piglets were found to be PPgV RNA negative.

Chapter 3

50

Figure 3-4. PPgV viral genome quantity in serum (RNA positive results only) during

the course of infection in domestic pigs. Sampling periods of each animal, gestation of

animals A and B, and piglet sampling are indicated below. Animals A-F were PPgV

genome positive at the first sampling time point (week 0). Animals A and B were not

sampled during the first four weeks of gestation. Negative control animals G-J were

genome negative at their respective first sampling time point. Animals E, F, H, I, and

J remained PPgV RNA negative for the duration of sampling, and animal G and piglet

P8 were PPgV genome positive only in sampling week 17 and week 36 (piglet

sampling directly after birth, before colostrum intake), respectively.

Chapter 3

51

Discussion

Since the discovery of PPgV in recent years, studies have shown that the virus is

widely distributed and RNA detection rates ranged from 0% in Serbia, Taiwan and

Switzerland in one study to 15.1% in another study from the United States (Kennedy

et al., 2019, Yang et al., 2018). Though PPgV infections can persist, genome detection

alone may lead to underestimation of virus distribution, as animals with transient and

past infections can be overlooked (Baechlein et al., 2016, Dawson et al., 1996).

However, an assay for the detection of Abs induced during PPgV infection, which

would aid not only in the estimation of PPgV exposure, but also give deeper insights

into the immune response, has not been described so far. Therefore, in this study, PPgV

proteins were expressed intracellularly in E. coli to allow for the detection of PPgV-

specific Abs using Western blot and indirect ELISA.

The helicase domain of PPgV NS3 was expressed in the soluble cytoplasmic fraction

of E. coli, which facilitated the purification with IMAC under native conditions. Purity

of the protein was optimal at 40 mM Imidazole during washing and binding and

yielded elution with high protein concentrations. NS3h has a predicted protein size of

33.5 kDa, which coincides with results obtained here showing NS3h in Western blot at

a size of ~37 kDa.

PPgV NS3h-specific Abs were detectable in porcine sera using Western blot, showing

that PPgV NS3 can induce an Ab response in the porcine host. Results obtained here

indicate that viremia and NS3h Ab response can be detected simultaneously, similar

to observations in EPgV (Lyons et al., 2014). WB with purified NS3h protein evidenced

unspecific bands, as they can also be seen in Coomassie blue stained SDS-PAGE gel

(Figure 3-1), in one of 18 samples shown here (lane 6 in Figure 3-2). All other serum

samples reacted only with NS3h, suggesting that protein purity is sufficient for the

methods implemented here.

Chapter 3

52

Comparison of WB and ELISA results indicates that samples showing a clear band in

WB also have high ODs in ELISA. Nonetheless, ODs over 0.93 in ELISA250 were found

in serum samples that did not detect a clear NS3h-specific band in WB. So far, it is

unclear whether such high ELISA reactivities may be caused by ELISA background

(compare lane 2 in Figure 3-2) or whether WB background conceals NS3h-specific

bands. To yield better differentiation between Ab positive and negative samples,

ongoing experiments target further improvement of ELISA and WB conditions to

reduce background caused by serum components binding to ELISA plates and WB

membranes unspecifically, such as changing washing conditions (i.e. higher

concentrations of Tween20). Furthermore, E2t appears to be a promising immunogenic

PPgV protein for implementation in indirect Ab ELISA. The necessity of measuring

anti-E2 Abs, specifically those that are protein conformation-dependent, is evident in

HPgV serology (Berg et al., 1999, Tanaka et al., 1998, McLinden et al., 2006, Pilot-Matias

et al., 1996b, Tacke et al., 1997b). Experiments pertaining to the expression of soluble

E2t or purification of E2t under denaturing conditions followed by refolding are

ongoing.

This study also investigated possible transmission routes of PPgV. Analysis of PPgV

genome in nasal discharge, saliva, urine and feces showed that these are not probable

routes of virus shedding from persistently or transiently infected pigs. Likewise,

vaginal swab samples of animals A and B taken during the birth of their piglets did

not contain PPgV RNA, though animal A was viremic at the time, indicating that

transmission during birth may be unlikely. However, detection of PPgV RNA in the

serum of piglet P8 directly after birth suggests intrauterine infection. Sows were

housed separately for birth of piglets and P8 was born of sow B, which cleared viremia

before day 69 of gestation, making cross contamination of the piglet sample highly

unlikely. Intrauterine infection of P8 likely took place before day 69 of the gestation,

when PPgV RNA was detectable in serum of the sow. Clearance of the virus from

serum of P8 by week 38 (at 16 days of age) could have been facilitated by maternally

Chapter 3

53

derived Abs. None of the other piglets were found PPgV RNA positive for up to 10

weeks of age, suggesting that transmission from sow A, which was viremic at the time,

to piglets did not occur. The fact that this phenomenon was only found in one of 26

piglets, and that the Cq value was relatively high (37.61), indicates that intrauterine

infection is possible, but that the vertical transmission route is not efficient in pegivirus

infection of pigs. This coincides with higher PPgV genome detection rates found in

older animals than in piglets, as was seen in previous studies (Lei et al., 2019, Kennedy

et al., 2019). Blood-borne transmission between pigs could for instance take place

iatrogenically during vaccinations, drug injections or other treatments, through direct

blood contact of wounded pigs, or due to cannibalism.

Results obtained here show that PPgV is most probabaly not transmitted by virus

shedding in excretion, but that horizontal transmission by the blood-borne route and

occasional intrauterine transmission, as can be found in HPgV, are more likely

(Dawson et al., 1996, Linnen et al., 1996, Schmidt et al., 1996, Feucht et al., 1996). This

study also provides first insights into the PPgV-specific immune response by detection

of NS3 and E2-specific Abs in PPgV genome positive and negative porcine serum

samples. The establishment of reliable indirect ELISAs will allow for high throughput

Ab detection in serum samples and will increase the understanding of the immune

response induced during pegivirus infection. Furthermore, investigation of the Ab

presence in other species with improved test systems will give deeper insights into the

host range and elucidate possible reservoir hosts like wild boar, where PPgV genome

has not been detected so far (Kennedy et al., 2019).

Chapter 3

54

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Acknowledgements

We thank Vesna Milićević (Belgrade, Serbia), Ming-Chung Deng and Chia-Yi Chang

(New Taipei City, Taiwan), Serum Bank of the Federal Food Safety and Veterinary

Office (Bern, Switzerland), Animal and Plant Health Agency (Weybridge, UK),

Francesco Feliziani and Gian Mario De Mia (Perugia, Italy), Katarzyna Podgórska and

Katarzyna Stępniewska (Puławy, Poland), Hua-Ji Qiu and Yuan Sun (Harbin, China),

Sarah Derking and Jörg Tenhündfeld (Vreden, Germany) and Thomas Große Beilage

(Essen/Oldenburg, Germany) for providing porcine serum samples. Special thanks to

Alexander Postel and Inga Grotha for sample library preparation.

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4 Overall Discussion

4.1 PPgV RNA detection in domestic pig serum samples from Europe and

Asia

New high-throughput sequencing technologies are facilitating the rapid discovery of

ever more viral genome sequences, expanding our knowledge of the world’s virome

and presenting us with new viruses for exploration at an increasing rate.

Characterization of such newly found viruses is crucial for understanding their impact

on the host. At the beginning of this project, PPgV had only recently been discovered

and the information that was available was limited to three full-length genomic

sequences, RNA detection rate of 2.2% found in 255 German domestic pigs, and

observation of persistent infection in three pigs (Baechlein et al., 2016). Consequently,

this project aimed at broadening our knowledge of pegivirus infection in the porcine

host through the investigation of virus distribution, tissue tropism, transmission

routes, and antibody response.

Firstly, the establishment of a reliable qRT-PCR, combined with the creation of an in

vitro-transcribed RNA copy standard, was necessary for the detection and accurate

quantification of PPgV genome. Dual-labeled-probe-based qRT-PCR using TaqMan

polymerase (TM qRT-PCR) is a highly specific PCR method, that evidenced efficiency

of over 96% as measured using the in vitro-transcribed RNA copy standard in this

study (Kennedy et al., 2019). Additionally, no lack in sensitivity was observed when

comparing the newly developed TM qRT-PCR and the previously described SYBR-

Green-based qRT-PCR (Baechlein et al., 2016), indicating that sensitivity is not

impaired due to higher specificity caused by the addition of a probe. Moreover, partial

NS3 sequences acquired in the study after detection of PPgV by TM qRT-PCR (see

Chapter 4.3) evidenced up to three mismatches in the probe sequence (one of which

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was at the third position from the 5’-end), suggesting sufficient sensitivity of TM qRT-

PCR even with a certain degree of genetic diversity in the target viral RNA sequence.

Investigation of serum samples originating from apparently healthy domestic pigs

from Europe and Asia revealed that PPgV viremia was present in 2.7% of animals,

though the range covered 0% (Switzerland, Serbia, Taiwan) to 10.3% (Great Britain)

(Kennedy et al., 2019). As mentioned in Chapter 2, these detection rates coincide with

findings of PPgV RNA in pigs from Germany (2.2%) and China (7.25%), and are

somewhat lower than the rate found in USA (15.1%) (Baechlein et al., 2016, Lei et al.,

2019, Yang et al., 2018, Kennedy et al., 2019). Due to the apparently wide distribution

of PPgV, it seems unlikely that the virus is not present in the pig populations of

Switzerland, Serbia and Taiwan. The absence of detectable PPgV may rather be

attributable to limited sample size combined with low prevalence, and limitations of

the methods used here (Kennedy et al., 2019). Follow-up investigation of additional

samples, as well as Ab detection, will clarify whether PPgV may be present in pigs in

these countries. To date, the reasons for variance in the detection rates of PPgV RNA

between the different countries are unknown. The findings presented in this project

highlight that PPgV distribution within different age groups is uneven, increasing

from piglets to sows and boar, as was also reported for pigs in China (Kennedy et al.,

2019, Lei et al., 2019). This shows that factors like herd management could influence

PPgV spread within and between herds. Future research targeting virus entry into pig

populations and infection dynamics within herds is necessary to understand the

spread of PPgV.

4.2 Phylogenetic analyses of PPgV

At the beginning of this project, three full-length genome sequences were available

from PPgV-infected domestic pigs from Germany, which occupied a separate branch

in the phylogenetic tree of pegiviruses (Baechlein et al., 2016). In addition, three further

Chapter 4

61

near full-length PPgV sequences from pigs from the United States, which shared high

nucleotide identitities among them (96.2-97.0%) and somewhat lower identities with

sequences from Germany (83.7-89.0%), were reported (Yang et al., 2018). These six

sequences were included in phylogenetic analyses using a partial NS3-coding genome

region in this project, which additionally analyzed PPgV originating from Germany,

Italy, Poland, Great Britain and China. Clusters of sequences from geographically close

regions, as well as identical sequences obtained from pigs of the same herd indicated

local spread of PPgV (Kennedy et al., 2019). Additionally, global virus spread was

indicated by high similarity of sequences obtained from geographically distant regions

of the world (i.e. from China and Great Britain), and might be facilitated by trade of

pigs or pig products. As is seen in pegivirus infection of horses (Pegivirus D and E) and

humans (Pegivirus C and H), distinct pegivirus species can be found within the same

host species (Smith et al., 2016). Similarly, further pegivirus species that can infect pigs

but are distinct from Pegivirus K may so far be undiscovered.

In comparison to the relatedness of Pegivirus A isolates originating from bats and

primates, sequences clustering with PPgV (Pegivirus K) have solely been isolated from

pigs, which indicates specific host tropism (Baechlein et al., 2016, Kennedy et al., 2019,

Lei et al., 2019, Yang et al., 2018, Smith et al., 2016).

4.3 No detection of PPgV RNA in wild boar

In our study, 800 wild boar serum samples from Lower Saxony, Germany, tested

negative for PPgV RNA. As mentioned in Chapter 2, future investigation of wild boar

serum in Ab assays will help in determining whether wild boar are indeed negligible

as possible (reservoir) hosts for porcine pegiviruses. However, shared infection of wild

boar and domestic pigs can be seen in other members of the Flaviviridae, like CSFV and

Atypical porcine pestivirus, and also in other viruses like African swine fever virus or

Pseudorabies virus (Cagatay et al., 2018, Postel et al., 2018, Moennig, 2015, Guinat et

Chapter 4

62

al., 2016, Ruiz-Fons et al., 2008). Transmission routes of viruses between domestic pigs

and wild boar include direct contact of infected animals, but also indirect transmission

via inanimate vectors, for instance by the consumption of contaminated feed (Ruiz-

Fons et al., 2008, Guinat et al., 2016, Fritzemeier et al., 2000, Moennig, 2015). As

mentioned in Chapter 3 and further discussed in Chapter 4.6, PPgV transmission may

require contact with PPgV-infected blood or products containing such blood or blood

components, possibly reducing the probability of viral transmission between wild

boar and domestic pigs in the case of PPgV.

In any case, HPgVs have been suggested to be ancient viruses, as they are well-adapted

to their host, are widely distributed (even among highly isolated populations in

humans), with evidence of co-migration, and may well be a symbiont or commensal

of humans (Chivero and Stapleton, 2015, Simmonds and Smith, 1999, Pavesi, 2001,

Sharp and Simmonds, 2011). Furthermore, bats may be ancient hosts of pegiviruses,

according to the phylogenetic diversity of pegiviruses found in the different species

from diverse parts of the world (Quan et al., 2013). These are indicators that

pegiviruses could be present in the wild boar population. Due to hygiene control

measures implemented in pig production and to reasons mentioned above,

transmission of pegiviruses between wild boar and domestic pigs may be limited, and

pegiviruses in wild boar may by genetically distant. Consequently, wild boar

pegiviruses may be undetectable by RNA detection methods used in this study

(Kennedy et al., 2019). Once again, Ab assays may be a useful tool in the further

investigation of pegivirus infection in different wild boar populations.

4.4 Persistent and transient PPgV infections

Persistence of pegivirus infection in pigs could be shown here for five, six, and nine

months (animals A, B, and D in Chapter 3, see Figure 3-4), and was observed for 22

months in a previous study (Baechlein et al., 2016). These observations coincide with

Chapter 4

63

findings in HPgV infection, in which immunocompetent individuals typically clear

viremia within two years (Gutierrez et al., 1997, Tanaka et al., 1998).

One longitudinal study (sampling time points several months to years apart) described

HPgV viral load (VL) dynamics with increased VL directly from onset, steady VL level

during persistence, and no reduction in VL in the years preceding clearance of viremia,

though one individual showed a VL reduction in the months prior to clearance (Lefrere

et al., 1999). The observations made in this project with PPgV viremia, as seen in

Chapter 3, largely coincide with this description. However, VL increase and decrease

during onset and clearance, respectively, were evident within four weeks in this study

(Chapter 3, Figure 3-4, PCR negative to steady VL in animal D, and steady VL to PCR

negative in animals A, B, and D). These phenomena in PPgV infection might have

remained undetected, had samples been acquired less frequently. Therefore, sampling

every two to three weeks, as was performed in this project, was adequate to obtain

detailed insights into VL dynamics.

In the context of first investigations, the highest PPgV genome load in serum was

detected in a sample from Germany (2 × 106 copies / ml), and the average genome load

overall was lower for PPgV (3.8 × 105 copies / ml) than for HPgV (average plasma

concentration > 1 × 107 copies / ml) (Kennedy et al., 2019, George et al., 2003, Chivero

et al., 2014). However, higher genome loads were detected later (maximum 1.1 × 107

copies / ml; Chapter 3, Figure 3-4), thus reaching serum concentrations closer to those

observed in individuals infected with HPgV and similar to findings reported for EPgV

infection (George et al., 2003, Chivero et al., 2014, Kapoor et al., 2013a). In this project,

transient infection was evident in animal G, and may also have been the case in

animals C, E and F (Chapter 3, Figure 3-4). Intriguingly, the mean VL was higher in

persistently infected animals (2.4 × 106 copies / ml) than in those with apparently

transient infection (4.0 × 103 copies / ml). This shows that PPgV seems to establish an

efficient persistent infection, with constantly high viral replication at an, as of now,

Chapter 4

64

unknown replication site (possibly PBMCs, liver or thymus; see Chapter 4.5). Animals

A-F originated from the same herd and were of the same age group. Moreover, they

appear to have been infected with the same PPgV strain, according to preliminary

PPgV genome sequencing results, in which sequences acquired from animals A-F in

week 0 were identical (sequencing was performed as described in Chapter 2 (Kennedy

et al., 2019)). This indicates no clear tendency towards host or viral factors contributing

to this distinction. Persistent infections were also observed in EPgV infection in two

horses over 3.5 years, while four other horses apparently cleared viremia (Kapoor et

al., 2013a). Factors contributing to pegivirus persistence in immunocompetent hosts

are largely unknown. Concordantly, why certain animals clear PPgV infection

immediately, while others become persistently infected, at least for some time, remains

to be elucidated.

Here, one piglet was found PPgV RNA positive directly after birth, before colostrum

intake, indicating an intrauterine infection that took place before day 69 of the

gestation, while the sow was viremic. This strongly suggests that the piglet was

persistently infected for the remainder of the gestation after initial infection, and that

it cleared the infection within two weeks of birth, perhaps due to uptake of maternally

derived neutralizing Abs (Chapter 3).

Furthermore, short transient infections with detectable viremia for only days, as was

seen in animal G in Chapter 3, may be common in the porcine host, thus leading to

lower RNA detection rates. In HPgV, RNA and E2-Ab detection rates are added to

calculate the total exposure, because E2-specific Abs coincide with viral clearance

(Tacke et al., 1997, Gutierrez et al., 1997, Thomas et al., 1998). Should this also be the

case in PPgV infection, RNA detection alone would clearly underestimate PPgV

exposure. Future research should include Ab detection for examination of

seroprevalence among pig populations to resolve this possible issue.

Chapter 4

65

4.5 Investigation of PPgV tissue tropism

Determination of the tissue tropism is an essential step for better understanding the

pathogenesis and viral life cycle of PPgV. Detection of PPgV RNA in PBMCs (Chapter

2, Figure 2-2) by FISH and qRT-PCR shows parallels to HPgV cell tropism (Fogeda et

al., 1999, George et al., 2003, Chivero and Stapleton, 2015, Kennedy et al., 2019).

Moreover, the detection of PPgV in the thymus of infected animals coincides with

lymphotropism. It would be intriguing to investigate whether the thymus may be a

primary site of PPgV replication, and if so, whether age-related thymic involution

leads to a reduction in viral replication or to a shift in the replication site.

In this project, PPgV RNA detection in PBMCs, liver and thymus may be explained by

infection of hematopoietic precursor cells. It has been speculated that the primary

permissive cell type(s) for HPgV may be hematopoietic stem cells that carry the virus

throughout differentiation, leading to reduced activation and proliferation of

lymphocytes (Chivero and Stapleton, 2015, Chivero et al., 2014).

As has been found in HPgV, serum concentrations of PPgV RNA are higher than those

detected within organs, including the liver (Pessoa et al., 1998, Kennedy et al., 2019).

HPgV was initially thought to be hepatotropic, and indeed viral RNA was present in

the liver of infected patients, but further research indicated that the virus did not in

fact replicate in the liver (Chivero and Stapleton, 2015, Fan et al., 1999, Pessoa et al.,

1998, Tucker et al., 2000, Berg et al., 1999, Laskus et al., 1997, Laras et al., 1999). PPgV

RNA was detectable in the liver of three pigs, which could indicate hepatotropism, but

could also be attributed to accumulation of viral RNA in the liver and virus replication

within lymphocytes present in liver tissue, as mentioned above. HPgV has been

detected in the brain tissue of patients with encephalitis and it was suggested that the

virus may cross the blood-brain barrier passively in lymphocytes (Balcom et al., 2018,

Chapter 4

66

Tuddenham et al., 2019). Similar modes of passive transportation of PPgV to various

tissues may take place within infected PBMCs (Kennedy et al., 2019).

Findings described in Chapter 2 indicate that PBMCs are promising candidates for the

further exploration of PPgV replication sites (Kennedy et al., 2019). Analysis of PPgV

replication in PBMCs from infected animals ex vivo or infection of PBMCs from PPgV

RNA negative animals may result in enhanced understanding of the life cycle of

pegiviruses in pigs. Likewise, detection of negative-strand RNA in samples acquired

during the course of this project may facilitate the clarification of replication sites for

PPgV.

4.6 Insights into PPgV transmission routes

At present, little is known about the transmission of animal pegiviruses. HPgV has

been shown to be transmitted by exposure to infected blood, sexually, and vertically

from mother to child (Bhanich Supapol et al., 2009, Hino et al., 1998, Kleinman, 2001,

Lin et al., 1998, Ohto et al., 2000, Stapleton, 2003). Similarly, transmission of EPgV was

successful by inoculation of naïve animals with virus-containing serum, and the virus

was present in commercially available horse sera (Chandriani et al., 2013, Postel et al.,

2016).

In HPgV infection, RNA and Ab detection rates increase together with the probability

of infection from healthy blood donors in developed countries, to blood donors in

developing countries, and finally to high-risk groups (i.e. intravenous drug users),

where exposure rates (RNA plus Ab positive rate) can be nearly universal. PPgV was

detected in 0% - 10.3% in this study, and up to 15.1% in another study from the United

States, which may indicate that RNA and Ab detection rates are similar to those of

HPgV in healthy blood donors rather than individuals of high-risk groups.

Chapter 4

67

Results obtained in the presented study reveal that PPgV is probably not transmitted

by shedding of the virus by excretion (Chapter 3). Rather, as PPgV RNA is found most

abundantly in serum of infected animals, blood-borne horizontal transmission, sexual

transmission and transmission from sow to piglet appear more likely. As was shown

by the detection of PPgV RNA in one piglet directly after birth, before colostrum

intake, intrauterine infection appears to be possible, though maybe ineffective. This

suggests that horizontal transmission may be more prominent. Sexual transmission

from boar to sows may be possible during artificial insemination, while transmission

in both directions could occur during natural mating. Close contact and poor health

condition of pigs may increase the probability of blood-borne PPgV transmission, and

pig typical behavior, such as cannibalism, which can be induced by stress and high

animal density, may also contribute. PPgV particles or infectious microvesicles

containing PPgV genome, as they have been described in HPgV (Chivero et al., 2014),

may accumulate in the blood of infected animals and could facilitate blood-borne

transmission. Further evaluation of the above-mentioned PPgV transmission routes is

necessary and will aid in the understanding of PPgV infection dynamics within herds.

4.7 Antibody reactivity in Western blot and ELISA

As mentioned above, PPgV Ab detection will be a helpful tool for the further

investigation of prevalence in pig populations, host tropism and dynamics of the

immune response in persistent and transient infections, as well as of the relevance of

different antibody types and maternally derived Abs. First insights into serum Ab

reactivity were obtained in this project, indicating that PPgV NS3 and E2 are viable

candidates for the establishment of such assays (Chapter 3).

Mammalian expressed HPgV E2 was the preferable antigen for HPgV-specific Ab

detection (Dille et al., 1997), and HPgV E2-specific Abs become detectable coinciding

with clearance of viremia, are long-lasting, and provide a certain degree of protection

Chapter 4

68

from reinfection (Gutierrez et al., 1997, Tacke et al., 1997, Tanaka et al., 1998, Thomas

et al., 1998). In comparison, EPgV-specific Abs were detected using bacterially

expressed NS3h, and over 58% of horses tested in one study evidenced Ab reactivity

with this antigen (Lyons et al., 2014). Based on these previous reports, in this project,

NS3h and C-terminally truncated E2 were produced in E. coli and evaluated for their

reactivity with porcine serum Abs (Chapter 3).

High purity of PPgV NS3h was achieved by purification with IMAC in FPLC and

limits background induced by unspecific bacterial proteins in Western blot and ELISA

assays. As E2t was expressed in the insoluble cytoplasmic fraction (in bacterial

inclusion bodies), purification under denaturing conditions followed by refolding of

the protein are currently ongoing. Alternatively, alterations in bacterial expression and

production of soluble E2t (i.e. induction at lower temperatures or expression in

bacterial strains specifically designed for production of soluble protein) may aid in

purification of the protein in its native form.

Although a lack of detectable human serum Abs against HPgV antigens other than E2

has been described (McLinden et al., 2006, Pilot-Matias et al., 1996a, Pilot-Matias et al.,

1996b, Fernandez-Vidal et al., 2007), preliminary results obtained here show that PPgV

NS3h is immunogenic. Multiple PPgV RNA positive and negative porcine serum

samples evidenced reactivity with this antigen. Results found here for PPgV NS3h

coincide with evidence of equine serum Ab reactivity observed with EPgV NS3h

(Lyons et al., 2014).

Comparison of Ab reactivity with PPgV NS3h versus with E2t is interesting and will

shed light on which roles these antigens play in the immune response of pigs infected

with PPgV. Porcine serum samples acquired here will aid in determining the dynamics

of the immune response induced in PPgV infection, and it will be interesting to discern

whether detection of E2t-specific Abs coincides with clearance of viremia, as is seen in

HPgV infection (Gutierrez et al., 1997, Tacke et al., 1997, Tanaka et al., 1998, Thomas et

Chapter 4

69

al., 1998). Reinfection, or reoccurrence of viremia, was observed in one animal

(Chapter 3, animal D). A possible explanation for this observation includes the

production of neutralizing Abs leading to viral clearance, followed by a drop in Ab

levels thus being insufficient to prevent reinfection. Alternatively, it is possible that

PPgV remained within the animal though viremia was cleared or under the detection

limit of the TM qRT-PCR. In either case, this is the first description of such a reinfection

(or reoccurrence of viremia) event for PPgV infection. In HPgV, dropping of E2-Ab

levels and subsequent reinfection have been described, and such causal relations

between Ab levels and reinfection may also be plausible for PPgV (Devereux et al.,

1998). The circumstances under which PPgV infection is eliminated need to be further

studied, including the specific mechanisms of the humoral and cellular immune

response regarding clearance of the virus from the replication site and from blood.

Efforts for the validation of NS3- and E2-based Ab detection assays are currently

ongoing and offer a promising step towards expanding our knowledge of pegivirus

infections in pigs. Firstly, this will provide information on the seroprevalence of PPgV

in pig populations from different countries, including those in which PPgV RNA has

not been detected to date, which will give a more accurate estimation of the exposure

of pigs to this recently discovered virus. Secondly, Ab presence in wild boar and in

additional potential host species will give insights into the host tropism of Pegivirus K

and into possible reservoir hosts. Lastly, dynamics and properties of the humoral

immune response induced in PPgV infection can be determined using such Ab assays.

An additional, enormous advantage for the further characaterization of PPgV would

be the ability to grow the virus in cell culture. Difficulties in culturing of HPgV have

been described, and to date, the best results are obtained using primary human PBMCs

(Chivero and Stapleton, 2015). As mentioned above, the usefulness of porcine PBMCs

will need to be evaluated.

Chapter 4

70

In conclusion, the results obtained in this project provide important first insights into

the biological characterisitics of PPgV, including the virus distribution, tissue tropism,

infection dynamics, and transmission routes, as well as the host immune response.

Future studies should focus on evaluating the impact that this recently discovered and

widely distributed virus has on porcine health.

Summary

71

5 Summary

Title: Biological characterization of porcine pegivirus

Author: Johanna Kennedy

Porcine pegivirus (PPgV) is a recently discovered member of the genus Pegivirus

within the Flaviviridae family. The genus comprises a group of enveloped, positive-

sense, single-stranded RNA viruses that can cause persistent infections in their hosts,

but, thus far, appear to be mostly apathogenic.

In this project, the investigation of domestic pig serum samples from Europe and Asia

(n = 1,736) revealed a wide distribution of the virus and presence of PPgV RNA in 2.7%

of animals and 15.2% of pig herds. In contrast, PPgV genome was not detectable in

serum samples originating from wild boar hunted in Lower Saxony, Germany (n =

800). Phylogenetic analyses of a highly conserved genome region within PPgV non-

structural protein 3 (NS3) revealed pairwise nucleotide identities > 90% within PPgV

sequences, and suggest that PPgV is spread locally, but can also be disseminated across

long distances.

In tissue samples of PPgV-viremic pigs, viral RNA was found most abundantly in the

liver, thymus and in peripheral blood mononuclear cells (PBMCs), not only by qRT-

PCR, but also using fluorescence in situ hybridization. These results indicate hepato-

and lymphotropism, and suggest that further investigations of possible replication

sites of PPgV should focus on PBMCs and hepatocytes as possible candidates.

Close examination of the PPgV genome content in serum and excretion samples

obtained from PPgV-positive pigs and their piglets longitudinally over the course of

11 months showed that shedding of the virus via excretion routes is unlikely, and that

intrauterine infection appears to be possible, though it may represent a rather

Summary

72

ineffective form of transmission. Instead, PPgV may be transmitted most effectively

horizontally via the blood-borne or sexual routes, similar to human pegiviruses.

As assays for the detection of PPgV-specific antibodies have not been described to

date, in this project, partial PPgV NS3 and E2 proteins were expressed in E. coli and

evaluated for their suitability in the establishment of an antibody-detection assay.

Preliminary results indicate the reactivity of porcine serum antibodies with both

proteins. The establishment and validation of an antibody enzyme-linked

immunosorbent assay are currently ongoing.

Taken together, the results obtained during the course of this project provide valuable

insights into the epidemiology and biological properties of porcine pegivirus infection,

and the methods developed here will aid in the further characterization of this newly

discovered and widely distributed virus.

Zusammenfassung

73

6 Zusammenfassung

Titel: Biologische Charakterisierung des porzinen Pegivirus

Autor: Johanna Kennedy

Das porzine Pegivirus (PPgV) wurde erst kürzlich identifiziert und gehört zum Genus

Pegivirus der Familie Flaviviridae. Das Genus umfasst behüllte, positiv-strängige RNA-

Viren, die persistente Infektionen verursachen können, aber in den meisten Fällen

apathogen zu sein scheinen.

In diesem PhD-Projekt wurde PPgV RNA in Serumproben von Hausschweinen aus

Europa und Asien (n = 1.736) bei 2,7% der Einzeltiere und in 15,2% der Bestände

nachgewiesen, was die weite Verbreitung des Virus verdeutlicht. Hingegen konnte

das Virus nicht in Serumproben von Wildschweinen aus Niedersachsen in

Deutschland (n = 800) detektiert werden. Phylogenetische Analysen konservierter

Genomregionen des PPgV Nicht-Strukturprotein 3 (NS3) zeigten innerhalb der PPgV

Sequenzen paarweise Nukleotididentitäten von über 90% und lassen vermuten, dass

PPgV sowohl regional als auch über weite Distanzen verbreitet werden kann.

Die höchsten Konzentrationen an PPgV RNA wurden mittels qRT-PCR und

Fluorezenz-in-situ-Hybridisierung in Leber- und Thymusgewebe und in peripheren

mononukleären Blutzellen (PBMCs) virämischer Tiere nachgewiesen. Diese

Ergebnisse deuten auf einen Leber- bzw. Lymphtropismus hin. Aus diesem Grund

bieten sich PBMCs und Leberzellen als vielversprechende Kandidaten für die

zukünftige Untersuchung der Replikation von PPgV an.

Die Untersuchung von Serum und Ausscheidungen PPgV-positiver Schweine und

ihrer Ferkel über einen Zeitraum von 11 Monaten hinweg zeigte, dass das Virus

höchstwahrscheinlich nicht aktiv ausgeschieden wird. Intrauterine Infektionen

Zusammenfassung

74

scheinen möglich, wenn auch wenig effektiv zu sein. Stattdessen wird PPgV

höchstwahrscheinlich eher horizontal über Blut und venerisch übertragen, wie es auch

beim humanen Pegivirus der Fall ist.

Testverfahren zum Nachweis PPgV-spezifischer Antikörper wurden bislang nicht

beschrieben. Aus diesem Grund wurden in diesem Projekt Teile des PPgV NS3-

Proteins und des Hüllproteins E2 in E. coli exprimiert und deren Verwendbarkeit für

die Etablierung eines serologischen Verfahrens untersucht. Erste Ergebnisse deuten

darauf hin, dass porzine Serumantikörper beide Proteinen binden können. Die

Etablierung und Validierung eines PPgV-Antikörper-ELISA sind Bestandteile aktuell

laufender Experimente.

Im Laufe dieses PhD-Projekts wurden wertvolle Einsichten in die Epidemiologie und

die biologischen Eigenschaften der PPgV-Infektion gewonnen, und die hier

entwickelten Methoden werden die weitergehende Charakterisierung dieses neu

entdeckten und weit verbreiteten Virus vorantreiben.

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Acknowledgements

Acknowledgements

First of all, I would like to thank Prof. Paul Becher, Prof. Karl-Heinz Waldmann and

Prof. Eike Steinmann for their excellent supervision and the very helpful supervisor

meetings during the course of my PhD project, and for the opportunity of conducting

this work at the Institute of Virology. Additionally, I want to thank Dr. Imke Steffen

for stepping in to evaluate this thesis.

I want to mention especially Christine Bächlein, PhD – your door was always open

and your support in all matters was endless – thank you!

I want to thank all my colleagues for teaching me in the ways of the virologists, and

for their input during many valuable discussions. Special thanks to Inga Grotha, Doris

à Wengen and Ester Barthel, who never failed to provide help with the unending

searches for samples, equipment and answers. Thanks to Dr. Alexander Postel and Dr.

Denise Meyer for many ideas and helpful comments. I would also like to thank Regina

Behre, as well as Prof. Beatrice Grummer, Dr. Tina Selle and Tanja Czeslik from the

HGNI, for their kindness and helpfulness in all PhD matters and the organization of

congresses and meetings.

Thanks to the staff from the Clinic for Swine and Small Ruminants of the TiHo,

especially Dr. Doris Höltig and Klaus Schlotter, and all others who cared for the pigs

that were part of this project and who acquired samples. Additionally, thanks to the

colleagues from the Institute of Pathology, Dr. Vanessa Maria Pfankuche, PhD,

Malgorzata Ciurkiewicz, PhD, Dr. Florian Hansmann, PhD, and Prof. Wolfgang

Baumgärtner, for their expertise in pathology and for conducting fluorescence in situ

hybridization.

I want to thank our late colleague, Prof. Ludwig Haas, who was not only my favorite

professor during vet school, but who inspired me to choose this career path.

Acknowledgements

Thanks also to Joe Brannan for proofreading – I hope that your somewhat random

knowledge of pegiviruses will come in handy one day.

A huge thanks goes to all my friends, who made these past years special (and the

downfalls bearable) through shared laughter, frustration and coffee – in particular

Kirsten Hülskötter, Dr. Lena Baron, Franziska Holzapfel, Gökce Nur Cagatay, PhD,

Nele Gremmel and Dr. Oliver Suckstorff. You made our little office on the 5th floor the

coffee-haven that it is, and the past years would not have been the same without you.

Lastly, I want to thank my whole family and especially my parents, Micaela Kennedy

and Jens Kunze, for their never-ending support and patience, my brother, Dr. Ricardo

Kennedy, for creating footsteps to follow in, and Lars Reise, for his constant love,

compassion and understanding.